Expression of Mammalian Proteins in Pseudomonas Fluorescens

ABSTRACT

The invention is a process for improved production of a recombinant mammalian protein by expression in a Pseudomonad, particularly in a  Pseudomonas fluorescens  organism. The process improves production of mammalian proteins, particularly human or human-derived proteins, over known expression systems such as  E. coli  in comparable circumstances. Processes for improved production of isolated mammalian, particularly human, proteins are provided.

REFERENCE TO PRIOR APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/230,192, filed Aug. 5, 2016, which is a continuation of U.S.application Ser. No. 11/400,840, filed Apr. 7, 2006, now U.S. Pat. No.9,458,487, granted Oct. 4, 2016, which is a continuation of U.S.application Ser. No. 11/038,901, filed Jan. 18, 2005, now U.S. Pat. No.9,453,251, granted Sep. 27, 2016, which claims priority to both of U.S.Provisional Application Nos. 60/564,798, entitled “Expression ofMammalian Proteins in Pseudomonas fluorescens,” filed Apr. 22, 2004, and60/537,148, entitled “Protein Expression Systems,” filed Jan. 16, 2004;U.S. application Ser. No. 11/038,901 is also a continuation-in-part ofU.S. application Ser. No. 10/681,540, entitled “Amended RecombinantCells for the Production and Delivery of Gamma Interferon as anAntiviral Agent, Adjuvant And Vaccine Accelerant,” filed Oct. 7, 2003,now U.S. Pat. No. 7,338,794, granted Mar. 4, 2008, which in turn claimspriority to U.S. Provisional Application No. 60/417,124, filed Oct. 8,2002.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created June 21, 2018, isnamed 38194-703.303_SL.txt and is 34,657 bytes in size.

FIELD OF THE INVENTION

The invention is a process for improved production of a recombinantmammalian protein by expression in a Pseudomonad, particularly in aPseudomonas fluorescens organism. The process improves production ofmammalian protein expression over known expression systems.

BACKGROUND OF THE INVENTION

More than 325 million people worldwide have been helped by the more than155 biotechnology drugs and vaccines approved by the U.S. Food and DrugAdministration (FDA). In addition, there are more than 370 biotech drugproducts and vaccines currently in clinical trials targeting more than200 diseases, including various cancers, Alzheimer's disease, heartdisease, diabetes, multiple sclerosis, AIDS, and arthritis. Unliketraditional small molecule therapeutics that are produced throughclassical chemical synthesis, proteins are usually produced in livingcells inefficiently and at high cost. Due to the high cost andcomplexity, there is a shortage of manufacturing capacity forprotein-based therapeutics.

The use of microbial cells to produce products has a very long history.As early as 1897, Buchner discovered that enzymes extracted from yeastare effective in converting sugar into alcohol, leading to theproduction of key industrial chemicals using microorganisms. By the1940s, large-scale production of penicillin via fermentation wasachieved. Techniques for the insertion of foreign genes into bacteriawere first developed in the early 1970s. Bacterial production ofcommercially viable recombinant mammalian protein was first exploited inthe production of human insulin (Goeddel, et al., 1979a; Wong, 1997).Today fermentation and cell culture underlie the bulk of the industry'sproduction of alcohol, antibiotics, biochemicals and therapeuticproteins. However, development and manufacturing of therapeuticallyuseful proteins has been hampered due, in large part, to the limitationsof the current organisms used to express these exogenous proteins.

Prokaryotic vs. Eukaryotic Protein Expression

Although bacterial expression system are often used to producerecombinant eukaryotic proteins, typically the proteins yielded differsignificantly from their original counterparts. In general, it is achallenge to reproduce the eukaryotic secondary and tertiary structuresin E. coli expression systems. At the same time, while the eukaryoticexpression systems currently are better able to form the secondary andtertiary structures of recombinant eukaryotic proteins, the capacity ofthese systems to produce recombinant proteins in large quantity islimited.

Post-translational modifications represent the most significantdifferences between prokaryotic and eukaryotic protein expression.Prokaryotes (i.e., bacteria) have a very simply cellular structure andno membrane-bound organelles. In eukaryotes, a protein is often modifiedafter it is initially produced. These modifications, in many cases, arenecessary to convert the peptide into a functional form. Thus, even whenexisting bacterial expression systems produce a protein with the correctprimary structure, the protein may not be post-translationally modifiedand is therefore often nonfunctional. Common modifications includedisulfide bond formation, glycosylation, acetylation, acylation,phosphorylation, and gamma-carboxylation, all of which can regulateprotein folding and biological activity. Bacterial expression systemsgenerally do not properly glycosylate, acetylate, acylate,phosphorylate, or gamma-carboxylate eukaryotic proteins.

Bacteria, such as E. coli, can form disulfide bonds, but the bonds areoften formed in the incorrect configuration required for biologicalactivity; therefore, denaturation and refolding is usually required toproduce active eukaryotic proteins. Molecular chaperone proteins arepresent in both prokaryotes and eukaryotes that facilitate the foldingof other proteins. In the absence of such chaperones, unfolded orpartially folded polypeptide chains are unstable within the cell,frequently folding incorrectly or aggregating into insoluble complexes.The binding of chaperones stabilizes these unfolded polypeptides,thereby preventing incorrect folding or aggregation and allowing thepolypeptide chain to fold into its correct conformation. However,chaperones differ in each type of cell, and can be differentiallyexpressed based on extracellular conditions.

Problems With Current Expression Systems

Escherichia coli (E. coli) is the most widely and routinely used proteinexpression system. Production in E. coli is inexpensive, fast, and wellcharacterized. Further, scale-up and harvesting is possible and cGMPproduction is well established. However, there are significantlimitations to the use of E. coli, which often prove difficult toovercome, particularly when expressing recombinant mammalian proteins.

Along with the limitations described above, the high-level expression ofrecombinant gene products in E. coli often results in the misfolding ofthe protein of interest and its subsequent degradation by cellularproteases or deposition into biologically inactive aggregates known asinclusion bodies. Protein found in inclusion bodies typically must beextracted and renatured for activity, adding time and expense to theprocess. Typical renaturation methods involve attempts to dissolve theaggregate in concentrated denaturant, and subsequent removal of thedenaturant by dilution. Some of the factors which have been suggested tobe involved in inclusion body formation include the high localconcentration of protein; a reducing environment in the cytoplasm (E.coli cytoplasm has a high level of glutathione) preventing formation ofdisulfide bonds; lack of post-translational modifications, which canincrease the protein solubility; improper interactions with chaperonesand other enzymes involved in in vivo folding; intermolecularcross-linking via disulfide or other covalent bonds; and increasedaggregation of folding intermediates due to their limited solubility. Itis probably a combination of these factors, as well as a limitedavailability of chaperones, which most commonly lead to the formation ofinclusion bodies.

Yeast expression systems, such as Saccharomyces cerevisiae or Pichiapastoris, are also commonly used to produce proteins. These systems arewell characterized, provide good expression levels, and are relativelyfast and inexpensive compared to other eukaryotic expression systems.However, yeast can accomplish only limited post-translational proteinmodifications, the protein may need refolding, and harvesting of theprotein can be a problem due to the characteristics of the cell wall.

Insect cell expression systems have also emerged as an attractive, butexpensive, alternative as a protein expression system. Correctly foldedproteins that are generally post-translationally modified can sometimesbe produced and extracellular expression has been achieved. However, itis not as rapid as bacteria and yeast, and scale-up is generallychallenging.

Mammalian cell expression systems, such as Chinese hamster ovary cells,are often used for complex protein expression. This system usuallyproduces correctly folded proteins with the appropriatepost-translational modifications and the proteins can be expressedextracellularly. However, the system is very expensive, scale-up is slowand often not feasible, and protein yields are lower than in any othersystem.

Pseudomonas fluorescens (P. fluorescens)

Pseudomonas fluorescens encompasses a group of common, nonpathogenicsaprophytes that colonize soil, water and plant surface environments. P.fluorescens are extensively used in agricultural and industrialprocesses, including commercially for the production of non-mammalianindustrial and agricultural proteins. Nonmammalian enzymes derived fromP. fluorescens have been used to reduce environmental contamination, asdetergent additives, and for stereoselective hydrolysis. Mycogen beganexpressing recombinant bacterial proteins in P. fluorescens in themid-1980's and filed its first patent application on the expression ofthe Bacillus thuringiensis toxin in P. fluorescens on Jan. 22, 1985(“Cellular encapsulation of biological pesticides”). Between 1985 and2004, Mycogen, later Dow Agro Sciences, as well as other companies,capitalized on the agricultural use of P. fluorescens in patentapplications on the production of pesticidal, insecticidal, andnematocidal toxins, as well as on specific toxic sequences and geneticmanipulation to enhance expression of these. Examples of patentapplications directed to the expression of recombinant bacterialproteins in P. fluorescens include: U.S. Pat. Nos. 3,844,893; 3,878,093,4,169,010; 5,292,507; 5,558,862; 5,559,015; 5,610,044; 5,622,846;5,643,774; 5,662,898; 5,677,127;5,686,282; 3,844,893; 3,878,093;4,169,010; 5,232,840; 5,292,507; 5,558,862; 5,559,015;5,610,044;5,622,846; 5,643,774; 5,662,898; 5,677,127; 5,686,282;5,686,283; 5,698,425; 5,710,031; 5,728,574;5,731,280; 5,741,663;5,756,087; 5,766,926; 5,824,472; 5,869,038; 5,891,688; 5,952,208;5,955,348;6,051,383; 6,117,670; 6,184,440; 6,194,194; 6,268,549;6,277,625; 6,329,172; 6,447,770; as well as PCT Publication Nos. WO00/15761; WO 00/29604; WO 01/27258; WO 02/068660; WO 02/14551; WO02/16940; WO 03/089455; WO 04/006657; WO 04/011628; WO 87/05937; WO87/05938; WO 95/03395; WO 98/24919; WO 99/09834; and WO 99/53035.

On Oct. 8, 2003, Dow AgroSciences filed PCT Publication No. 04/087864entitled, “Amended Recombinant Cells (ARCs) for the Production andDelivery of Antiviral Agents, Adjuvants and Vaccine Accelerants”. Theapplication describes recombinant cells that can include at least oneheterologous gene encoding a chemokine or a cytokine and theadministration of such cells to a host to accelerate an immune response.The application demonstrates the production of bovine interferon-α andinterferon-γ in P. fluorescens.

Dow Global Technologies currently has several pending patentapplications in the area of use of P. fluorescens to produce recombinantproteins. PCT Application WO 03/068926 to Dow Global Technologies, filedFeb. 13, 2003, entitled, “Over-Expression of Extremozyme Genes inPseudomonas and Closely Related Bacteria” describes an expression systemin which pseudomonads, specifically P. fluorescens, can be used as hostcells for the production of extremozyme enzymes. These enzymes aretypically ancient, found in prokaryotes, eukaryotes including fungi,yeast, lichen, protists and protozoa, algae and mosses, tardigrades andfish. The patent discloses that enzymes can be derived from certainextremophilic fungi and yeast, but are typically derived fromextremophilic bacteria.

PCT publication No. WO 03/089455 to Dow Global Technologies, filed Apr.22, 2003, entitled “Low-Cost Production of Peptides” describes a methodof producing small peptides, primarily antimicrobial peptides, asconcatameric precursors in Pseudomonads, specifically P. fluorescens.

PCT publication No. WO 04/005221 to Dow Global Technologies, entitled“Benzoate and Antranilate Inducible Promoters” provides novel benzoate-or anthranilate-inducible promoters from P. fluorescens, as well asnovel tandem promoters, variants and improved mutants thereof, that areuseful for commercial prokaryotic fermentation systems.

U.S. Pat. No. 5,232,840 to Monsanto Co. describes the use of novelribosomal binding sites to enhance expression of certain proteins inprokaryotic cells. In one example, the cells are used to express porcinegrowth hormone in several organisms, including E. coli, P. fluorescens,and P. putida. The data shows that P. fluorescens is less efficient atexpressing the growth hormone when compared to E. coli. In contrast,when expressing a bacterial protein, P. fluorescens is much moreeffective at protein production than E. coli under comparableconditions. In fact, P. fluorescens cells described in this patentproduce several-fold more bacterially-derived β-galactosidase than E.coli (compare table 4 to tables 1 and 2).

While progress has been made in the production of proteins of commercialinterest, a strong need remains to improve the capability and productionlevel of recombinant mammalian, and in particular human, proteins.

Therefore, it is an object of the present invention to provide a processfor the production of recombinant mammalian, in particular human,proteins that can be isolated and purified for therapeutic use, andcells which can accomplish this process.

It is a further object of the present invention to provide improvedprocesses for the production of active recombinant mammalian proteins,including complex mammalian proteins.

It is a further object of the present invention to provide improvedprocesses for the production of high levels of recombinant mammalian, inparticular human, proteins.

It is a further object of the present invention to provide transformedorganisms that provide high expression levels of soluble or insolublerecombinant mammalian proteins.

SUMMARY OF THE INVENTION

It has been discovered that Pseudomonas fluorescens is a superiororganism for the production of recombinant proteins, and in particularrecombinant mammalian proteins, such as recombinant human proteins.Based on these discoveries, the present invention provides a process ofproducing recombinant mammalian or mammalian-derived proteins in P.fluorescens. In addition, the invention provides P. fluorescenstransformed to produce recombinant mammalian, including human, proteins.

In one embodiment, the invention provides a process of producing amammalian protein in a P. fluorescens organism in which the protein isproduced at a higher level or concentration per cell or per liter offermentation reaction than in an E. coli organism under comparableconditions. In yet another embodiment, the invention provides a processof producing mammalian proteins in an P. fluorescens organism in a batchculture which produces higher amounts of protein per liter than acorresponding batch of recombinant E. coli organisms.

Comparable conditions or substantially comparable conditionsparticularly refers to expression of recombinant protein using the sameoperably linked transcriptional promoter and ribosomal binding site indifferent organisms, and using the same initial induction conditions.Comparable conditions can further include using the same vector andassociated regulatory elements, including, but not limited to, enhancersequences, termination sequences, and origin or replication sequences.Comparable conditions can also include the same total volume of cellfermentation reaction. Comparable conditions can also include the sameconcentration of total cells per liter of reaction. In one embodiment,the conditions also include total induction times (before measurement)that are similar or the same. However, in another embodiment, theinduction times can vary depending on the organism. Specifically, P.fluorescens has a capacity for increased growth time over E. coliwithout reducing protein production, such that protein production can bemeasured in P. fluorescens at a time point at which E. coli cells arelargely silent. One way to measure the comparable conditions is tocompare the percentage of recombinant protein per total cell protein.The comparable conditions also do not require identical media forgrowth. The media can be adjusted to ensure optimal production for theindividual organisms.

In another embodiment, the invention provides a process for producingrecombinant mammalian proteins by producing the proteins in a P.fluorescens organism and isolating the produced protein. In onesub-embodiment, the process includes substantially purifying theprotein. In one embodiment, the protein is derived from a human protein,or is humanized.

The invention also provides the use of P. fluorescens in at least thefollowing embodiments:

-   -   (i) the production of recombinant mammalian, including human,        proteins present in the cell in a range of between 1 and 75        percent total cell protein (% tcp), or in particular, at least        greater than approximately 5% tcp, 10% tcp, at least 15% tcp, at        least 20% tcp or more;    -   (ii) the production of recombinant mammalian, including human,        proteins that are soluble and present in the cytoplasm of the        cell in a range of between 1 and 75% tcp or in particular, at        least greater than approximately 5% tcp, 10% tcp, at least 15%        tcp, at least 20% tcp or more;    -   (iii) the production of recombinant mammalian, including human,        proteins that are insoluble in the cytoplasm of the cell, in a        range of between 1 and 75% tcp or in particular, at least        greater than approximately 5% tcp, 10% tcp, at least 15% tcp, at        least 20% tcp or more;    -   (iv) the production of recombinant mammalian, including human,        proteins that are soluble in the periplasm of the cell in a        range of between 1 and 75% tcp or in particular, at least        greater than approximately 5% tcp, 10% tcp, at least 15% tcp, at        least 20% tcp or more;    -   (v) the production of recombinant mammalian, including human,        proteins that are insoluble in the periplasm in a range of        between 1 and 75% tcp or in particular, at least greater than        approximately 5% tcp, 10% tcp, at least 15% tcp, at least 20%        tcp or more;    -   (vi) the production or recombinant mammalian, including human,        proteins in the cell in a range of between 1 and 75% tcp, or        particularly at least greater than approximately 5% tcp, 10%        tcp, at least 15% tcp, at least 20% tcp or more, when grown at a        cell density of at least 40g/L;    -   (vii) the production of recombinant mammalian, including human,        proteins present in the cell in an active form;    -   (viii) the production of multi-subunit recombinant mammalian,        including human, proteins in active form;    -   (ix) the production of recombinant mammalian, including human,        proteins that are then isolated and purified; and    -   (x) the production of recombinant mammalian, including human,        proteins that are renatured.

In one embodiment, the recombinant mammalian protein is selected fromthe group consisting of a multi-subunit protein, a blood carrierprotein, an enzyme, a full length antibody, an antibody fragment, or atranscriptional factor.

In another embodiment, the invention includes:

-   -   (i) Pseudomonas fluorescens organisms that are transformed to        produce recombinant mammalian, including human, proteins at a        higher level or concentration than a corresponding E. coli        organism when grown under substantially corresponding        conditions;    -   (ii) Pseudomonas fluorescens organisms that are transformed to        produce recombinant mammalian, including human, proteins and        peptides that are present in the cell in a range of between 1        and 75% tcp or in particular, at least greater than        approximately 5% tcp, 10% tcp, at least 15% tcp, at least 20%        tcp or more;    -   (iii) Pseudomonas fluorescens organisms that are transformed to        produce recombinant mammalian, including human, proteins that        are present in the cell in active form;    -   (iv) Pseudomonas fluorescens organisms that are transformed to        produce recombinant mammalian, including human, proteins that        are soluble in the cytoplasm of the cell in a range of between 1        and 75% tcp or in particular, at least greater than        approximately 5% tcp, 10% tcp, at least 15% tcp, at least 20%        tcp or more;    -   (v) Pseudomonas fluorescens organisms that are transformed to        produce recombinant mammalian, including human, proteins that        are insoluble in the cytoplasm of the cell in a range of between        1 and 75% tcp or in particular, at least greater than        approximately 5% tcp, 10% tcp, at least 15% tcp, at least 20%        tcp or more;    -   (vi) Pseudomonas fluorescens organisms that are transformed to        produce recombinant mammalian, including human, proteins that        are soluble in the periplasm of the cell in a range of between 1        and 75% tcp or in particular, at least greater than        approximately 5% tcp, 10% tcp, at least 15% tcp, at least 20%        tcp or more;    -   (vii) Pseudomonas fluorescens organisms that are transformed to        produce recombinant mammalian, including human, proteins that        are insoluble in the periplasm of the cell in a range of between        1 and 75% tcp or in particular, at least greater than        approximately 5% tcp, 10% tcp, at least 15% tcp, at least 20%        tcp or more;    -   (viii) Pseudomonas fluorescens organisms that are transformed to        produce multi-subunit recombinant mammalian, including human,        proteins;    -   (ix) Pseudomonas fluorescens organisms that are transformed to        produce multi-subunit recombinant mammalian, including human,        proteins present in the cell in active form.

In an alternative embodiment, a Pseudomonas organisms and closelyrelated bacteria other than fluorescens are used as host cells in thisinvention, as described in more detail below. In one embodiment, thehost cell will be selected generally from the genus Pseudomonas andspecifically from a nonpathogenic Pseudomonas species. Likewise, anyPseudomonas fluorescens strain can be used that accomplishes the desiredinventive goal, including but not limited to strain MB101, or a strainthat is modified to include at least one host-cell-expressible, insertedcopy of at least one Lac repressor protein-encoding lad transgene, suchas MB214 and MB217. The Pseudomonas organism can also optionally begenetically modified to add or delete one or more genes to improveperformance, processing, or other characteristics.

In one embodiment, the Pseudomonas organism is transformed with anucleic acid encoding a recombinant mammalian protein selected from thegroup consisting of a multi-subunit protein, a blood carrier protein, anenzyme, a full length antibody, an antibody fragment, or atranscriptional factor. In one embodiment, the P. fluorescens organismexpresses a recombinant mammalian protein selected from the groupconsisting of a multi subunit protein, a blood carrier protein, anenzyme, a full length antibody, an antibody fragment, or atranscriptional factor.

The expressed recombinant mammalian or human protein will typically havea mass of at least about 1 kD, and up to about 100, 200, 300, 400 or 500kD, often between about 10 kD and about 100 kD, and usually greater thanabout 30 kD.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graph showing hu-γ-IFN purified from the soluble fraction ofP. fluorescens samples displays activity comparable to a commerciallyavailable standard.

FIG. 2 is a picture of an ELISA showing the activity of purified Gal13in P. fluorescens and E. coli.

FIGS. 3A-3B represents human growth hormone expression constructs. Theamino acid sequence of human growth hormone lacking its native secretionsignal sequence is shown in FIG. 3A. Plasmid constructs for expressionin P. fluorescens (pDOW2400) and E. coli (412-001.hGH) are shown in FIG.3B.

FIGS. 4A-4B is a picture of an SDS-PAGE analysis of soluble andinsoluble fractions of hGH expressed in P. fluorescens and E. coli. Thetime post-induction is denoted by I0, I24, I48 (FIG. 4A), 0 or 3 (FIG.4B). The large arrows indicate the position of the 21 kDa hGH protein.

FIGS. 5A-5D shows an SDS-PAGE analysis of the expression of γ-IFN in E.coli versus P. fluoresens cells. Soluble (S) and insoluble (I) fractionsof samples taken at 0, 3, 24 and 48 hours post-induction (TO, etc.) wereresolved. E. coli expressed γ-IFN is shown in panel FIG. 5A, P.fluorescens expressed γ-IFN is shown in panel FIG. 5B. 5 μL of A575-20samples were loaded onto a 10% Bis-Tris NuPAGE gel and resolved in 1×MES. Arrows indicate the position of the recombinant protein. Westernanalyses are shown in FIG. 5C (E. coli) and FIG. 5D (P. fluorescens).

FIG. 6 shows the replacement of the BuiBui toxin gene with the BGI geneat the SpeI and XhoI sites of pMYC1803.

FIG. 7 shows that all the transformants selected had the desiredinterferon insert, as verified by sequencing the inserted DNA. Thesequence of the inserted DNA (SEQ ID NO: 17) encodes an amino acidsequence (SEQ ID NO: 29).

FIG. 8 represents the nucleotide sequence for the phosphate bindingprotein-gal2 single chain antibody fusion protein. The sequence of theDNA (SEQ ID NO: 18) encodes an amino acid sequence (SEQ ID NO: 19)

FIG. 9 represents the amino acid sequence for the phosphate bindingprotein-gal2 single chain antibody fusion protein.

FIG. 10 represents the nucleotide sequence for the phosphate bindingprotein-human growth hormone fusion protein. The sequence of the DNA(SEQ ID NO: 20) encodes an amino acid sequence (SEQ ID NO: 21).

FIG. 11 represents the amino acid sequence for the phosphate bindingprotein-human growth hormone fusion protein.

DETAILED DESCRIPTION OF THE INVENTION Process for Producing RecombinantMammalian Proteins

The invention provides processes and transformed Pseudomonas fluorescensorganisms that produce recombinant mammalian proteins.

In one embodiment, the invention provides a process for producingrecombinant mammalian proteins by producing the proteins in a P.fluorescens organism and isolating the produced protein. The protein canbe isolated after expression by techniques known in the art, including,but not limited to, affinity chromatography, ion-exchangechromatography, antibody affinity, size-exclusion, or any other methodthat eliminates a substantial portion of the cellular debris from theprotein. In one sub-embodiment, the process provides a substantiallypurified protein. The isolated protein can have activity similar to thatof the native protein that it is derived from. The protein can beisolated in a correctly folded state or conformation, approximating thatof the native protein, or can be further renatured or modified to put itinto a correctly folded conformation. In one sub-embodiment, the proteinis derived from a human protein, or is humanized. A “humanized” proteinis typically a chimeric mammalian-type protein which is partiallycomprised of a human-derived protein sequence. Humanization isparticularly useful in antibody production and the development ofhumanized antibodies has been extensively described, for example in U.S.Pat. No. 6,800,738.

In one embodiment, expression of the protein by the host cell isfollowed by isolation of the protein. In another embodiment, the proteinof peptide is purified. In an alternative embodiment, the protein ispurified following isolation of the protein. Optionally the isolated orpurified protein can be renatured or refolded in order to produce activeproteins.

In another embodiment, the invention provides a process of producing amammalian protein in a P. fluorescens organism in which the protein isproduced at a higher level or concentration than in an E. coli organism.The suitability of P. fluorescens organisms for high level production ofmammalian proteins was unexpected based on the lack of success inproducing such proteins in these organisms in the prior art. The presentinventors have found that these organisms are indeed capable of highlevels of production of mammalian proteins, and typically expressprotein in higher yield or at higher levels than E. coli when tested incorresponding assays. In another embodiment, the invention provides aprocess of producing mammalian proteins in an P. fluorescens organism ina batch culture which produces higher amounts of protein per liter thana corresponding batch of recombinant E. coli organisms.

In some embodiments, processes are provided that include producingrecombinant mammalian, including human, multi-subunit proteins in activeform in P. fluorescens; producing recombinant mammalian blood carrierproteins, including human blood carrier proteins such as transferrin andalbumin in P. fluorescens; producing recombinant mammalian enzymes,including recombinant mammalian enzymes in active form in P.fluorescens; producing antibodies and antibody fragments, includingsingle chain antibodies and Fab fragments in P. fluorescens; andproducing recombinant mammalian, including human, transcriptionalfactors in P. fluorescens.

In one embodiment, the recombinant mammalian protein is produced as amultimer, or in a concatameric precursor, for example, in the form of atleast two small peptide (1-15 amino acids) units in tandem. In analternative embodiment, the recombinant mammalian protein is notproduced as a multimer, or in concatameric precursors, but instead isproduced as a single chain polypeptide.

Screening of Biomolecules

A separate embodiment of the present invention provides P. fluorescensorganisms in a process of screening libraries of mammalian biomoleculesto identify at least one that exhibits a desired activity or property.The P. fluorescens cells can be transformed with a number of mammalianderived nucleic acids for which testing is desired, producing a libraryof transformed host cells. Upon expression, polypeptides encoded by atleast some of the nucleic acids are produced for testing either incytoplasm or following recovery from the cell. Examples of activitiesand properties for which testing may be performed include: polypeptideexpression level; polypeptide stability; and biocatalytic activities andproperties. Illustrative examples of biocatalytic activities andproperties include: enzymatic activities; protein interactions/binding;protein stabilization; substrate usage; product formation; reactionconditions, such as pH, salinity, or reaction temperature; biocatalyticparameters for a given catalyzed reaction, such as Km and Vmax; andstability behavior, such as thermostability and biocatalyst half-life.The test results obtained may be used to selected library member(s) forfurther development.

Protein Expression

A key aspect of this invention is the expression of high levels ofrecombinant mammalian, for example human, proteins in a range of between1 and 75 percent total cell protein (% tcp) by expression in P.fluorescens organisms. The expressed proteins can be soluble orinsoluble while in the P. fluorescens cell. Such high levels of solubleor insoluble recombinant mammalian proteins can be an improvement overpreviously known mammalian protein expression systems. In particular,high levels of recovered mammalian proteins in large scale fermentationreactions are not typically possible with known techniques.

In one embodiment, the invention provides expression levels of mammalianproteins that exceed those found in E. coli expression systems. In oneembodiment, the concentration of recombinant protein in each cell ishigher than that found in E. coli in comparative assays. In oneembodiment, the level of recombinant protein as compared to total cellprotein measured in the P. fluorescens expression system is higher thanthat of the same recombinant protein expressed in E. coli. In anotherembodiment, the level or amount of soluble protein in the P. fluorescensexpression system described herein is higher than the level or amount ofsoluble recombinant protein in a comparable E. coli expression system.In another embodiment, the total amount of active protein is higher thanthe amount derived from an E. coli expression system. In a separateembodiment, the level of recombinant active protein as compared to totalcell protein measured in the P. fluorescens expression system is higherthan that of the same recombinant protein expressed in E. coli. In oneembodiment, the level, concentration, or amount of protein expressed inP. fluorescens is at least 2×, at least 3×, at least 4×, at least 5×, atleast 6×, at least 7×, at least 8×, at least 9×, at least 10×, or morethe level, concentration, or amount of recombinant protein expressed inE. coli in comparable assays.

One of the benefits of P. fluorescens as an expression system is thatthe cells can be grown in large scale cultures without negativelyimpacting their capacity for protein production. This capacity exceedsthat found in other bacterial systems, such as E. coli. In anotherembodiment, the process includes producing mammalian proteins in batchcultures in which the recombinant protein is produced at a higher totallevel in P. fluorescens than in E. coli batch cultures. In yet anotherembodiment, the invention provides a process of producing mammalianproteins in an P. fluorescens organism in a batch culture which produceshigher amounts of protein per liter than a corresponding batch ofrecombinant E. coli organisms.

The invention generally provides processes and transformed P.fluorescens organisms that afford expression levels of 1-75% total cellprotein (tcp) of soluble or insoluble recombinant mammalian proteins.The recombinant mammalian proteins expressed in the cell can beexpressed in an active form. In other embodiments, the P. fluorescensprovides at least 1, 5, 10, 15, 20, 25, 30, 40, 50, 55, 60, 65, 70, or75% tcp of recombinant mammalian proteins.

These proteins can be soluble, and when soluble, can be present in thecytoplasm or periplasm of the cell during production. Soluble proteinsare readily released from the cell by methods including, but not limitedto, rupturing of the cell membrane by pressure (i.e. the “French” pressmethod), or by lysozyme degradation of the cell membrane. Cells cantypically also be lysed using detergents, such as non-ionic detergents.Proteins that are soluble can be further stabilized by adjustingcomponents of the buffer, such as buffer pH, salt concentrations, oradditional protein components (for example, in multi-subunit complexes).The soluble proteins can be isolated or purified from other protein andcellular debris by, for example, centrifugation and/or chromatographysuch as size exclusion, anion or cation exchange, or affinitychromatography.

The proteins can also be insoluble. Insoluble proteins are typicallyfound in inclusion bodies in the cytoplasm, but are also often in theperiplasm. Not all insoluble proteins are in inclusion bodies, and canalso be found in membrane aggregates, as small protein aggregates or inany other insoluble form in the cytoplasm or periplasm. Insolubleproteins can typically be renatured using, for example, reducing agentssuch as urea or guanidine hydrochloride. Insoluble proteins or proteinaggregates can be isolated, for example, by centrifugation and/orchromatography such as size exclusion chromatography. Proteins ininsoluble aggregates can typically be separated by solubilization of theaggregates using, for example, micelles or reverse micelles as describedin Vinogradov, et al. (2003) Anal Biochem. 15; 320(2):234-8.

In a particular embodiment, the Pseudomonas host cell can have arecombinant mammalian peptide, polypeptide, protein, or fragment thereofexpression level of at least 1% tcp and a cell density of at least 40g/L, when grown (i.e. within a temperature range of about 4° C. to about55° C., inclusive) in a mineral salts medium. In a particularembodiment, the expression system will have a recombinant protein ofpeptide, including recombinant mammalian protein, expression level of atleast 5% tcp and a cell density of at least 40 g/L, when grown (i.e.within a temperature range of about 4° C. to about 55° C., inclusive) ina mineral salts medium at a fermentation scale of at least 10 Liters.

Expression levels can be measured by standard techniques known in theart. In one embodiment, the amount of protein (in grams) is compared tothe amount in grams of total cell protein in a given sample. In anotherembodiment, the measurement is a level of recombinant protein per liter.In another embodiment, the level or amount can be measured as comparedto a known standard, such as a BSA controL The level or amount ofrecombinant protein can be measured, for example, by analyzing the lightabsorbtion of a purified protein, by measuring an affinity of an markerfor the protein (such as an antibody affinity) and comparing that to aknown standard, or by measuring the level of activity compared to aknown standard (such as a known amount of purified, active protein).

It has been found that, in certain situations, no additionaldisulfide-bond-promoting conditions or agents are required in order torecover disulfide-bond-containing target polypeptides in active, solubleform, when a Pseudomonas fluorescens bacteria is used as the expressionhost celL Therefore, in one embodiment, the transgenic peptide,polypeptide, protein, or fragment contains at least one intramoleculardisulfide bond in its native state. In other embodiments, the proteincan contain up to 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20 or moredisulfide bonds in its native state.

In some embodiments, the protein is expressed or found in the periplasmof the cell during production before purification or isolation. Theprotein can be secreted into the periplasm by being fused to anappropriate signal secretion sequence. In one embodiment, the signalsequence is a signal sequence that is native to the P. fluorescensgenome. In specific embodiments, the signal sequence is a phosphatebinding protein, a Lys-Arg-Orn binding protein (LAObp or KRObp)secretion signal peptide, an Outer Membrane Porin E (OpreE) secretionsignal peptide, an azurin secretion signal peptide, an iron (III)binding protein (Fe(III)bp) secretion signal peptide, or a lipoprotein B(LprB) secretion signal peptide.

In one embodiment, the recombinant peptide, polypeptide, protein, orfragment thereof has a folded intramolecular conformation in its activestate. P. fluorescens typically produce mammalian proteins moreefficiently in the correctly folded conformation. In one embodiment,more than 50% of the expressed, transgenic peptide, polypeptide,protein, or fragment thereof produced can be produced as singlepeptides, polypeptides, proteins, or fragments thereof in soluble,active form or insoluble, but renaturable form in the cytoplasm orperiplasm. In another embodiment about 60%, 70%, 75%, 80%, 85%, 90%, 95%of the expressed protein is obtained in or can be renatured into activeform.

Definitions

Throughout this specification, the term “protein” is used to include anyamino acid concatamers or polymers. The terms “polypeptide,” “peptide”and “protein” are used interchangeably and include amino acid polymersin which one or more amino acid residue is an artificial chemicalanalogue of a corresponding naturally occurring amino acid, as well asto naturally occurring amino acid polymers.

The term “isolated” refers to nucleic acid, protein, or peptide that issubstantially or essentially free from other material components whichnormally accompany it as found in its native state when in a cell, forexample, other cellular components.

The term “purified” or “substantially purified” is used to mean that theprotein is separated from other cell components and is separated fromother proteins and peptides found in the cell that are not in a nativecomplex with the protein. In particular embodiments, the purifiedproteins are of a purity approved for therapeutic or veterinary used asdefined by standard cGMP guidelines or approved by the FDA.

The term “percent total cell protein” (“tcp”) means the amount ofprotein in the host cell as a percentage of aggregate cellular protein.Alternatively, the term means a measure of the fraction of total cellprotein that represents the relative amount of a given protein expressedby the cell.

The term “operably attached” refers to any configuration in which thetranscriptional and any translational regulatory elements are covalentlyattached to the encoding sequence in such disposition(s), relative tothe coding sequence, that in and by action of the host cell, theregulatory elements can direct the expression of the coding sequence.

As used herein, the term “mammal” is meant to include or designate anyanimal in the class Mammalia including human or non-human mammals, suchas, but not limited, to porcine, ovine, bovine, rodents, ungulates,pigs, swine, sheep, lambs, goats, cattle, deer, mules, horses, monkeys,apes, dogs, cats, rats, and mice.

As used herein, the term “recombinant mammalian protein” or peptide ismeant to include proteins derived from a native mammalian proteinsequence or derived or generated from a native mammalian nucleic acidsequence. Such recombinant proteins can be produced from nucleic acidsequence substantially corresponding to native mammalian mRNA orsubstantially corresponding cDNA, or fragements thereof. The sequencecan be adjusted accordingly based on specific host cell codon usage asknown in the art.

The phrase “substantially corresponding” in the context of two nucleicacids or polypeptides refers to the residues in the two sequences thathave at least 50%, 60%, 70%, 80%, 90%, or higher identity when alignedfor maximum correspondence over a domain of the protein, as measuredusing an algorithms known in the art. Optimal alignment of sequences forcomparison can be conducted, e.g., by algorithms known in the art (e.g.Smith & Waterman (1981) Adv. Appl. Math. 2:482; Needleman & Wunsch(1970) J. Mol. Biol. 48:443; Pearson & Lipman (1988) Proc. Nat'l. Acad.Sci. USA 85:2444; Altschul et al. (1990) J. Mol. Biol. 215:403-410(BLAST)), by computerized implementations of these algorithms (GAP,BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package,Genetics Computer Group, 575 Science Dr., Madison, Wis.), or byinspection. Software for performing BLAST analyses is publicly availablethrough the National Center for Biotechnology Information(http://www.ncbi.nlm.nih.gov/).

The term “fragment” means a portion or partial sequence of a nucleotide,protein, or peptide sequence.

As used herein, the term “soluble” means that the protein is notprecipitated by centrifugation at between approximately 5,000× and20,000× gravity when spun for 10-30 minutes in a buffer underphysiological conditions. Soluble proteins are not part of an inclusionbody or other precipitated mass.

As used herein, the term “insoluble” means that the protein that can beprecipitated by centrifugation at between 5,000× and 20,000× gravitywhen spun for 10-30 minutes in a buffer under physiological conditions.Insoluble proteins can be part of an includion body or otherprecipitated mass.

The term “inclusion body” is meant to include any intracellular bodycontained within a cell wherein an aggregate of proteins have beensequestered.

As used herein, the term “homologous” means either i) a protein that hasan amino acid sequence that at least 70, 75, 80, 85, 90, 95, or 98%similar to the sequence of a given original protein and that retains adesired function of the original protein or ii) a nucleic acid that hasa sequence that is at least 70, 75, 80, 5, 90, 95, or 98% similar to thesequence of a given nucleic acid and that retains a desired function ofthe original nucleic acid sequence. In all of the embodiments of thisinvention and disclosure, any disclosed protein, peptide or nucleic acidcan be substituted with a homologous or substantially homologousprotein, peptide or nucleic acid that retains a desired function. In allof the embodiments of this invention and disclosure, when any nucleicacid is disclosed, it should be assumed that the invention also includesall nucleic acids that hybridize to the disclosed nucleic acid.

In one non-limiting embodiment, the non-identical amino acid sequence ofthe homologous polypeptide can be amino acids that are members of anyone of the 15 conservative or semi-conservative groups shown in Table 1.

TABLE 1 Similar Amino Acid Substitution Groups Conservative Groups (8)Semi-Conservative Groups (7) Arg, Lys Arg, Lys, His Asp, Glu Asn, Asp,Glu, Gln Asn, Gln Ile, Leu, Val Ile, Leu, Val, Met, Phe Ala, Gly Ala,Gly, Pro, Ser, Thr Ser, Thr Ser, Thr, Tyr Phe, Tyr Phe, Trp, Tyr Cys(non-cystine), Ser Cys (non-cystine), Ser, Thr

Types of Mammalian Proteins Produced

In general, the recombinant mammalian protein can be any mammalianprotein of which an amino acid sequence is known or any putativemammalian or mammalian-derived protein for which an amino acid sequenceis deduced. The proteins can be selected from the group consisting of amulti-subunit protein, a blood carrier protein, an enzyme, a full lengthantibody, an antibody fragment, or a transcriptional factor.

The amino acid sequence of the protein can be altered to adjust fordesired qualities, such as to ensure certain types of interactions. Thesequence can, for example, be adjusted to reduce immunoreactivity, or toincrease absorbtion, reduce excretion, or otherwise enhancebioavailability in an organism such as a mammaL The amino acid sequenceof the protein can thus be adjusted to ensure certain post-translationalmodifications or protein conformations.

In one embodiment, the mammalian protein is a chemokin or cytokine. Inanother embodiment, the mammalian proteins is one of the followingproteins: IL-1, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8,IL-9, IL-10, IL-11, IL-12, IL-12elasti, IL-13, IL-15, IL-16, IL-18,IL-18BPa, IL-23, IL-24, VIP, erythropoietin, GM-CSF, G-CSF, M-CSF,platelet derived growth factor (PDGF), MSF, FLT-3 ligand, EGF,fibroblast growth factor (FGF; e.g., aFGF (FGF-1), bFGF (FGF-2), FGF-3,FGF-4, FGF-5, FGF-6, or FGF-7), insulin-like growth factors (e.g.,IGF-1, IGF-2); tumor necrosis factors (e.g., TNF, Lymphotoxin), nervegrowth factors (e.g., NGF), vascular endothelial growth factor (VEGF);interferons (e.g., IFN-α, IFN-β, IFN-γ); leukemia inhibitory factor(LIF); ciliary neurotrophic factor (CNTF); oncostatin M; stem cellfactor (SCF); transforming growth factors (e.g., TGF-α, TGF-β1, TGF-β1,TGF-β1; TNF superfamily (e.g., LIGHT/TNFSF14, STALL-1/TNFSF13B (BLy5,BAFF, THANK), TNFalpha/TNFSF2 and TWEAK/TNFSF12); or chemokines(BCA-1/BLC-1, BRAK/Kec, CXCL16, CXCR3, ENA-78/LIX, Eotaxin-1,Eotaxin-2/MPIF-2, Exodus-2/SLC, Fractalkine/Neurotactin, GROalpha/MGSA,HCC-1, I-TAC, Lymphotactin/ATAC/SCM, MCP-1/MCAF, MCP-3, MCP-4,MDC/STCP-1/ABCD-1, MIP-1α, MIP-1β, MIP-2α/GROβ, MIP-3α/Exodus/LARC,MIP-3α/Exodus-3/ELC, MIP-4/PARC/DC-CK1, PF-4, RANTES, SDFlα, TARC, orTECK).

Alternatively, the protein is not a chemokine or cytokine. In anotherembodiment, the mammalian protein is not one of the following proteins:IL-1, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9,IL-10, IL-11, IL-12, IL-12e1asti, IL-13, IL-15, IL-16, IL-18, IL-18BPa,IL-23, IL-24, VIP, erythropoietin, GM-CSF, G-CSF, M-CSF, plateletderived growth factor (PDGF), MSF, FLT-3 ligand, EGF, fibroblast growthfactor (FGF; e.g., aFGF (FGF-1), bFGF (FGF-2), FGF-3, FGF-4, FGF-5,FGF-6, or FGF-7), insulin-like growth factors (e.g., IGF-1, IGF-2);tumor necrosis factors (e.g., TNF, Lymphotoxin), nerve growth factors(e.g., NGF), vascular endothelial growth factor (VEGF); interferons(e.g., IFN-α, IFN-β, IFN-γ); leukemia inhibitory factor (LIF); ciliaryneurotrophic factor (CNTF); oncostatin M; stem cell factor (SCF);transforming growth factors (e.g., TGF-α, TGF-β1, TGF-β1, TGF-β1); TNFsuperfamily (e.g., LIGHT/TNFSF14, STALL-1/TNFSF13B (BLy5, BAFF, THANK),TNFalpha/TNFSF2 and TWEAK/TNFSF12); or chemokines (BCA-1/BLC-1,BRAK/Kec, CXCL16, CXCR3, ENA-78/LIX, Eotaxin-1, Eotaxin-2/MPIF-2,Exodus-2/SLC, Fractalkine/Neurotactin, GROalpha/MGSA, HCC-1, I-TAC,Lymphotactin/ATAC/SCM, MCP-1/MCAF, MCP-3, MCP-4, MDC/STCP-1/ABCD-1,MIP-1α, MIP-1β, MIP-2α/GROβ, MIP-3α/Exodus/LARC, MIP-3β/Exodus-3/ELC,MIP-4/PARC/DC-CK1, PF-4, RANTES, SDF1α, TARC, or TECK). In oneembodiment, the protein is not a porcine protein, particularly not aporcine growth factor.

As yet a further embodiment of the present disclosure, the recombinantmammalian proteins, their fragments or other derivatives, or analogsthereof, can be antibodies. These antibodies can be, for example,polyclonal or monoclonal antibodies. This aspect of the presentinvention also includes chimeric, single chain, and humanizedantibodies, as well as Fab fragments, or the product of a Fab expressionlibrary.

Production of Multi-subunit Proteins

In one embodiment of the present invention, the production ofrecombinant mammalian multi-subunit proteins by a host cell of thespecies Pseudomonas is provided. In another embodiment, a host cell ofthe Pseudomonas species is provided that has been transformed to expressa recombinant mammalian multi-subunit protein. In one embodiment,multi-subunit proteins, including recombinant mammalian or humanproteins, are expressed in a Pseudomonas host celL In one embodiment,expression of the multi-subunit protein by the host cell is followed byisolation of the multi-subunit protein. In another embodiment, themulti-subunit protein of peptide is purified. The protein can beassembled by the cell before purification or isolation, or furtherassembly can be undertaken during or after isolation or purification.Optionally, the protein or any portion thereof can be renatured orrefolded to produce active proteins.

Any of a variety of vectors and expression systems can be used toexpress the multi-subunit protein in the host cell. The multi-subunitscan be located on a single vector, optionally operably linked todifferent promoters, optionally in a polycistronic sequence. Eachsubunit can also be on different vectors. Multiple vectors can be used.Each subunit can be under the control of one or more selection markers.Regulatory elements can be included on the vector, including periplasmicsecretion signal sequences, internal ribosome entry sites, activatorsequences, promoters, and termination signals.

In one embodiment, multisubunit proteins are expressed in Pseudomonasusing expression systems with auxotrophic selection markers as disclosedin U.S. application Ser. No. 10/994,138 to Dow Global Technologies filedNov. 19, 2004, wherein the control of each nucleic acid encoding asubunit is under the control of an auxotrophic selection marker.Multisubunit proteins that can be expressed include homomeric andheteromeric proteins. The multisubunit proteins may include two or moresubunits, that may be the same or different. For example, the proteinmay be a homomeric protein comprising 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, or more subunits. The protein also may be a heteromeric proteinincluding 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more subunits.

Exemplary multisubunit mammalian proteins include: receptors includingion channel receptors; signaling proteins such as kinases, GTPases,ATPases; transmembrane proteins; extracellular matrix proteins includingchondroitin; collagen; immunomodulators including MHC proteins, fullchain antibodies, and antibody fragments; enzymes including RNApolymerases, and DNA polymerases; and membrane proteins.

Production of Blood Proteins

In one embodiment of the present invention, the production ofrecombinant mammalian blood proteins is provided. In one embodiment,expression of the blood protein by the host cell is followed byisolation of the blood protein. In another embodiment, the blood proteinis purified. In another embodiment, following isolation of the bloodprotein, the blood protein is purified. Optionally, the protein can berenatured or refolded to produce active protein. In general, arecombinant blood protein of this invention is produced by transforminga suitable host cell, such as a P. fluorescens host cell, with a nucleicacid construct encoding the blood protein, culturing the transformedhost cell under conditions appropriate for expression, and optionallyisolating, or isolating and purifying the recombinant blood proteinexpressed by the cell.

In another embodiment, a host cell of the lPseudomonas species isprovided that has been transformed to express a recombinant mammalianblood protein with an vector containing appropriate genes and regulatoryelements for expression of the blood protein of interest is provided.

The blood proteins that can be expressed include, but are not limitedto: carrier proteins, such as albumin, including human albumin (Seq IDNo. 1, Table 2) and bovine albumin; transferrin, including humantransferrin (Seq ID No. 2, Table 2), bovine transferrin, rattransferrin, recombinant transferrin, recombinant transferrinhalf-molecules, recombinant transferrin half-molecules having alteredproperties; haptoglobin; fibrinogen and other coagulation factors;complement components; immunoglobulins; enzyme inhibitors; precursors ofsubstances such as angiotensin and bradykinin; insulin; endothelin;globulin including alpha, beta, and gamma-globulin; and other types ofproteins, peptides, and fragments thereof found primarily in the bloodof mammals. The amino acid sequences for numerous blood proteins havebeen reported (see, S. S. Baldwin (1993) Comp. Biochem Physiol. 106b:203-218), including the amino acid sequence for human serum albumin(Lawn, L. M., et al. (1981) Nucleic Acids Research 9:22; pp 6103-6114)and human serum transferrin (Yang, F. el al. (1984) Proc. Nat'l. Acad.Sci. USA 81; pp. 2752-2756).

In a specific embodiment, the production of albumin in P. fluorescens,is provided, comprising transforming a P. fluorescens host cell with anexpression vector containing a nucleic acid sequence or sequences andregulatory elements for expression of albumin, culturing the host cellunder conditions suitable for expression of the albumin, and recoveringthe albumin expressed by P. fluorescens. According to this embodiment,the albumin expressed is selected from the group consisting of humanalbumin, bovine albumin, rabbit albumin, chicken albumin, rat albumin,and mouse albumin. In another embodiment, albumin can be fused to atherapeutically active polypeptide, which can be of mammalian ornon-mammalian origin.

In a further specific embodiment, the production of a transferrin in P.fluorescens is provided, comprising transforming a P. fluorescens hostcell with an expression vector containing nucleic acid and regulatoryelements for expression of the transferrin, culturing the host cellunder conditions suitable for expression of the transferring. In anotherembodiment, following expression of the transferrin, and, in oneembodiment, isolating the protein. In a further embodiment, thetransferrin can be purified following isolation. The transferrinexpressed is selected from the group consisting of human serumtransferrin, glycosylated human transferrin, non-glycosylated humantransferrin, the N-terminal half-molecule of human transferrin, bovinetransferrin, rat transferrin, mouse transferrin, primate transferrin,recombinant transferrin, recombinant transferrin half-molecules,recombinant transferrin half-molecules having altered properties,transferrin polynucleotides, transferrin polypeptides encoded bytransferrin polypeptides, transferrin polypeptides, transferrinantibodies, transferrin fragments, and transferrin fused to atherapeutically active polypeptide.

In yet another specific embodiment, the production of a globulin in P.fluorescens is provided, comprising transforming a P. fluorescens hostcell with an expression vector containing nucleic acid and regulatoryelements for expression of the globulin, culturing the host cell underconditions suitable for expression of the globulin and optionallyisolating the protein. In a further embodiment, following expression,the globulin is isolated and purified from the host celL The globulinexpressed is selected from the group consisting of human globulin,bovine globulin, rabbit globulin, rat globulin, mouse globulin, sheepglobulin, monkey globulin, steroid-binding globulins, and globulin fusedto a therapeutically active polypeptide.

In a further embodiment, the production of an insulin in P. fluorescensis provided, comprising transforming a P. fluorescens host cell with anexpression vector containing nucleic acid and regulatory elements forexpression of the insulin, culturing the host cell under conditionssuitable for expression of the insulin and optionally isolatig theprotein. In a further embodiment, the insulin can be isolated andpurified following production of the insulin by the host celL Theinsulin expressed is selected from the group consisting of humaninsulin, bovine insulin, mouse insulin, rat insulin, porcine insulin,monkey insulin, and insulin fused to a therapeutically activepolypeptide. The accession number for human insulin genes is J00265, andfor synthetic human insulin gene the accession number is J02547.

Full-length DNA for production of recombinant blood proteins ortruncated DNA encoding either the amino-terminal or carboxy-terminallobe of blood proteins or a portion thereof can be obtained fromavailable sources or can be synthesized according to the known sequencesby standard procedures.

TABLE 2Sequences of blood proteins expressed by the system of the present disclosure.Amino Seq. ID. MKWVTFISLLFLFSSAYSRGVFRRDAHKSEVAHRFKDLGE Acid No: 1ENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVAD SequenceESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQE of humanPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFL serumKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKA albuminACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFKQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEE GKKLVAASQAALGL(Lawn, et al. (1981) Nuc. Ac. Rsch. 9(22):6103-6114) Amino Seq. ID.MRLAVGALLVCAVLGLCLAVPDKTVRWCAVSEHEATKC Acid No: 2QSFRDHMKSVIPSDGPSVACVKKASYLDCIRAIAANEADA SequenceVTLDAGLVYDAYLAPNNLKPVVAEFYGSKEDPQTFYYAV ofAVVKKDSGFQMNQLRGKKSCHTGLGRSAGWNIPIGLLYC transferrinDLPEPRKPLEKAVANFFSGSCAPCADGTDFPQLCQLCPGCGCSTLNQYFGYSGAFKCLKNGAGDVAFVKHSTIFENLANKADRDQYELLCLDNTRKPVDEYKDCHLAQVPSHTVVARSMGGKEDLIWELLNQAQEHEGICDKSICEFQLFSSPHGICDLLFKDSAHGFLKVPPRMDAKMYLGYEYVTAIRNLREGTCQEAPTDECKPVKWCALSHHERLKCDEWSVNSVGKIECVSAETTEDCIAKIMNGEADAMSLDGGFVYIAGKCGLVPVLAENYNKSDNCEDTPEAGYFAVAVVKKSASDLTWDNLKGKKSCHTAVGRTAGWNIPMGLLYNKINHCRFDEFFSEGCAPGSKKDSSLCKLCMGSGLNLCEPNNKEGYYGYTGAFRCLVEKGDVAFVKHQTVPQNTGGKNPDPWAKNLNEKDYELLCLDGTRKPVEEYANCHLARAPNHAVVTRKDKEACVHKILRQQQHLFGSNVTDCSGNFCLFRSETKDLLFRDDTVCLAKLHDRNTYEKYLGEEYVKAVGNLRKCSTSSLLEACTFRRP(Strausberg (2002) PNAS, 99:16899-16903) cDNA Seq. ID.atggccctgtggatgcgcctcctgcccctgctggcgctgctggccctctggggacctgacc SequenceNo: 3 cagccgcagcctttgtgaaccaacacctgtgcggctcacacctggtggaagctctctacctaof Human gtgtgcggggaacgaggcttcttctacacacccaagacccgccgggaggcagaggacctInsulin gcaggtggggcaggtggagctgggcgggggccctggtgcaggcagcctgcagcccttggccctggaggggtccctgcagaagcgtggcattgtggaacaatgctgtaccagcatctgctccctctaccagctggagaactactgcaactag

Production of Enzymes

In one embodiment of the present invention, the production ofrecombinant mammalian enzymes or co-factors by a host cell of thespecies Pseudomonas fluorescens is provided. In another embodiment, ahost cell of the Pseudomonas species is provided that has beentransformed to express a recombinant mammalian enzyme or co-factor.

The enzymes and co-factors expressed in this embodiment include but arenot limited to aldolases, amine oxidases, amino acid oxidases,aspartases, B₁₂ dependent enzymes, carboxypeptidases, carboxyesterases,carboxylyases, chemotrypsin, CoA requiring enzymes, cyanohydrinsynthetases, cystathione synthases, decarboxylases, dehydrogenases,alcohol dehydrogenases, dehydratases, diaphorases, dioxygenases, enoatereductases, epoxide hydrases, fumerases, galactose oxidases, glucoseisomerases, glucose oxidases, glycosyltrasferases, methyltransferases,nitrile hydrases, nucleoside phosphorylases, oxidoreductases,oxynitilases, peptidases, glycosyltrasferases, peroxidases, and enzymesfused to a therapeutically active polypeptide.

In another embodiment, the enzyme can be a mesophilic enzymepolypeptide, for example one that is desirable for human and/orveterinary therapeutic and/or diagnostic use. Examples of suchtherapeutic mesophilic enzymes include, e.g., tissue plasminogenactivator; urokinase, reptilase, streptokinase; catalase, superoxidedismutase; DNAse amino acid hydrolases (e.g., asparaginase,amidohydrolases); carboxypeptidases; proteases, trypsin, pepsin,chymotrypsin, papain, bromelain, collagenase; neuraminidase; lactase,maltase, sucrase, and arabinofuranosidases.

Yet another embodiment provides for the production of recombinant enzymereplacements in P. fluorescens cells by transforming a P. fluorescenshost cell with an expression vector containing nucleic acids andregulatory elements for xpression of recombinant enzyme replacements,and culturing the cell under conditions suitable for expression of therecombinant enzyme replacements. The recombinant enzyme replacementsexpressed in the host cell is selected from the group consisting ofAlgasidase beta, Laronidase, and recombinant enzyme replacements fusedto a therapeutically active polypeptide.

Production of Mammalian Antibodies and Antibody Fragments

In one embodiment of the present invention, the production ofrecombinant mammalian single chain, Fab fragments and/or full chainantibodies or fragments or portions thereof by a host cell of thespecies P. fluorescens is provided. In one embodiment, followingexpression of the protein, the protein can be isolated and optionallypurified. Optionally, the protein can be renatured to produce an activeprotein. The antibody or antibody fragments are optionally linked to asecretion signal sequence for targeting in the cell during production.

In another embodiment, a host cell of the Pseudomonas species isprovided that has been transformed to express a recombinant mammaliansingle chain, Fab fragments and/or full chain antibodies or fragments orportions thereof.

In one embodiment, the P. fluorescens cell can produces a single chainantibody or fragments or portions thereof. A single-chain antibody caninclude the antigen-binding regions of antibodies on a singlestably-folded polypeptide chain. Single-chain antibodies are of smallersize than classical immunoglobulins but can retain the antigen-specificbinding properties of antibodies. Single chain antibodies can be usedfor therapeutics, such as “naked” single-chain antibodies, bi-specificantibody binders, radioconjugates or as fusions with effector domains,diagnostics, such as tumor imaging or in vivo or ex vivo cancer markerassays, research tools, such as protein purification and detection,including identification and characterization of novel therapeutictargets, antibody microarrays, display technologies and/or vehicles forgene or drug delivery.

In another embodiment, the P. fluorescens cell produces Fab fragments orportions thereof. Fab fragments can be a piece of a particular antibody.The Fab fragment can contain the antigen binding site. The Fab fragmentcan contain 2 chains: a light chain and a heavy chain fragment. Thesefragments can be linked via a linker or a disulfide bond.

In other embodiments of the present invention, full chain antibodies canbe expressed in P. fluorescens, and other Pseudomonas species. An intactantibody containing the Fc region can be more resistant againstdegradation and clearance in vivo, thereby having longer biological halflife in circulation. Such antibodies can be used as a therapeutic agentfor diseases requiring sustained therapies.

In one embodiment, a process for producing a functional antibody orfragment thereof in Pseudomonas species is provided by providing anexpression vector that contains separate cistronic or polycistronicsequences. The separate cistron expression vector can contain a firstpromoter-cistron pair for expression of an immunoglobulin light chainand a second promoter-cistron pair for expression of an immunoglobulinheavy chain, such that expression of the light chain and heavy chain areindependently regulated by separate promoters. Each cistron within theexpression cassette polynucleotide can include a translation initiationregion (TIR) operably linked to the nucleic acid sequence coding for thelight chain or heavy chain of the full length antibody. In oneembodiment, the TIR sequences can be manipulated to provide differenttranslational strength combinations for light and heavy chains. In analternative embodiment, a heavy chain coding sequence can be located onthe same plasmid as a light chain coding sequence. In an alternativeembodiment, the heavy and light chain sequences are found in apolycistronic sequence within a single plasmid, or coded into the genomeof the host.

In another embodiment, a process is provided for producing a functionalantibody or fragment thereof in a host cell transformed with twoseparate translational units respectively encoding the light and heavychains of the antibody. In one embodiment the process includes: a)culturing the host cell under suitable conditions so that the lightchain and heavy chain are expressed in a sequential fashion, therebytemporally separating the production of the light and heavy chains; andb) allowing the assembly of the light and heavy chains to form thefunctional antibody or fragment thereof.

In further embodiment, the Pseudomonas expression system can expresshuman therapeutic single chain, Fab fragments or full chain antibodiesor portions thereof, including, but not limited to Fab, Fab′, F(ab′)₂,F(ab′)₂-leucine zipper, Fv, dsFv, anti-CD18 antibody, chimericantibodies, human antibodies, humanized antibodies, or those describedin the Table 3 below.

TABLE 3 Antibodies and Antibody Fragments. Product Antibody TargetAntigen Type Isotype Indication 5G1.1 Complement (C5) Humanised IgGRheumatoid Arthritis 5G1.1 Complement (C5) Humanised IgG SLE 5G1.1Complement (C5) Humanised IgG Nephritis 5G1.1-SC Complement (C5)Humanised ScFv Cardiopulmanory Bypass 5G1.1-SC Complement (C5) HumanisedScFv Myocardial Infarction 5G1.1-SC Complement (C5) Humanised ScFvAngioplasty ABX-CBL CBL Human GvHD ABX-CBL CD147 Murine IgG Allograftrejection ABX-IL8 IL-8 Human IgG2 Psoriasis AD-159 gp120 Humanised HIVAD-439 gp120 Humanised HIV Antegren VLA-4 Humanised IgG MultipleSclerosis Anti-CD11a CD11a Humanised IgG1 Psoriasis Anti-CD18 CD18Humanised Fab′2 Myocardial infarction Anti-LFA1 CD18 Murine Fab′2Allograft rejection Anti-VEGF VEGF Humanised IgG1 Cancer (general)Antova CD40L Humanised IgG Allograft rejection Antova CD40L HumanisedIgG SLE BEC2 anti-Id Murine IgG Lung BIRR-1 ICAM-1 Murine IgG2a StrokeBTI-322 CD2 Rat IgG GvHD C225 EGFR Chimeric IgG Head + Neck CAT-152TGF-beta 2 Human Glaucoma Surgery CDP571 TNF-alpha Humanised IgG4Crohn's CDP571 TNF-alpha Humanised IgG4 Rheumatoid Arthritis CDP850E-selectin Humanised Psoriasis Corsevin M Fact VII ChimericAnticoagulant D2E7 TNF-alpha Human Rheumatoid Arthritis HerceptinHer2/neu Humanised IgG1 Metastatic Breast HNK20 F gP Murine IgA RSVHu23F2G CD11/18 Humanised Multiple Sclerosis Hu23F2G CD11/18 HumanisedIgG Stroke IC14 CD14 — Toxic shock ICM3 ICAM-3 Humanised PsoriasisIDEC-114 CD80 Primatised Psoriasis IDEC-131 CD40L Humanised SLE IDEC-131CD40L Humanised Multiple Sclerosis IDEC-151 CD4 Primatised IgG1Rheumatoid Arthritis IDEC-152 CD23 Primatised Asthma/ Allergy InfliximabTNF-alpha Chimeric IgG1 Rheumatoid Arthritis Infliximab TNF-alphaChimeric IgG1 Crohn's LDP-01 beta2-integrin Humanised IgG Stroke LDP-01beta2-integrin Humanised IgG Allograft rejection LDP-02 alpha4beta7Humanised Ulcerative Colitis LDP-03/ CD52 Humanised IgG1 CLL Campath1HLym-1 HLA DR Chimeric NHL LympoCide CD22 Humanised NHL MAK-195F TNFalpha Murine Fab′2 Toxic shock MDX-33 CD64 (FcR) Human Autoimmunehaematogical disorders MDX-CD4 CD4 Human IgG Rheumatoid ArthritisMEDI-500 TCR alpha beta Murine IgM GvHD MEDI-507 CD2 Humanised PsoriasisMEDI-507 CD2 Humanised GvHD OKT4A CD4 Humanised IgG Allograft rejectionOrthoClone CD4 Humanised IgG Autoimmune disease OKT4A Orthoclone/ CD3Murine mIgG2a Allograft rejection anti-CD3 OKT3 Ostavir Hep B Human HepB OvaRex CA 125 Murine Ovarian Panorex 17- EpCAM Murine IgG2a Colorectal1A PRO542 gp120 Humanised HIV Protovir CMV Humanised IgG1 CMV RepPro/AbcgpIIbIIIa Chimeric Fab Complications of coronary iximab angioplastyrhuMab-E25 IgE Humanised IgG1 Asthma/Allergy Rituxan CD20 Chimeric IgG1NHL SB-240563 IL5 Humanised Asthma/Allergy SB-240683 IL-4 HumanisedAsthma/Allergy SCH55700 IL-5 Humanised Asthma/Allergy Simulect CD25Chimeric IgG1 Allograft rejection SMART a- CD3 Humanised Autoimmunedisease CD3 SMART a- CD3 Humanised Allograft rejection CD3 SMART a- CD3Humanised IgG Psoriasis CD3 SMART CD33 Humanised IgG AML M195 SMART HLA— NHL 1D10 Synagis F gP Humanised IgG1 RSV (Paediatric) VitaxinVNRintegrin Humanised Sarcoma Zenapax CD25 Humanised IgG1 Allograftrejection

Production of Transcriptional Factors

In one embodiment of the present invention, the production ofrecombinant mammalian transcription factors by a host cell of thespecies Pseudomonas fluorescens is provided. In one embodiment,following expression of the protein, the protein can be isolated. Inanother embodiment, the protein can be purified. Optionally, the proteincan be renatured to produce an active protein. In another embodiment, ahost cell of the Pseudomonas species is provided that has beentransformed to express a recombinant mammalian transcription factor.

Transcription factors suitable for insertion into the expression systemsof the present invention include those of the helix turn helix familyand members of the Pac family, as well as other transcription factorfamilies known in the art. Members of these families suitable for usewith the present invention include mammalian and mammalian homologs andanalogs of: transcriptional regulators; transcription factors of the ofthe ASNC family such as ASNC_trans_reg, putative transcriptionalregulators; bacterial regulatory proteins of the luxR family; bacterialregulatory helix-turn-helix transcription factors; bacterial regulatoryproteins of the arsR family; transcription factors of thehelix-turn-helix domain, especially the rpiR family; bacterialregulatory protein transcription factors, bacterial regulatoryhelix-turn-helix transcription factors; DNA binding domain transcriptionfactors; MarR family of transcription factors; the ROK family oftranscription factors; the MerR family of regulatory proteins; argininerepressor transcription factors; firmicute transcriptional factors;ferric uptake regulatory transcription factors; sigma transcriptionfactors; response regulatory receiver transcription factors; tryptophanRNA-binding attenuator protein transcription factors; putativesugar-binding domain transcription factors; PRD domain transcriptionfactors; nitrogen regulatory protein transcription factors; negativeregulators of genetic competence, such as MecA; negative transcriptionalregulator transcription factors; bacterial transcriptional regulatortranscription factors; glycerol-3-phosphate responsive transcriptionfactors; iron dependent repressor transcription factors; and numerousspecies specific transcriptional regulator transcription factors.

Transcriptional factors expressed by Pseudomonas species can be utilizedfor diagnostic, therapeutic, and investigational applications.

Vector Preparation Polynucleotides

The recombinant mammalian proteins and peptides can be expressed frompolynucleotides in which the target polypeptide coding sequence isoperably attached to transcription and translation regulatory elementsforming a functional gene from which the host cell can express theprotein. The coding sequence can be a native coding sequence for thetarget polypeptide, if available, but can also be a coding sequence thathas been selected, improved, or optimized for use in the selectedexpression host cell: for example, by synthesizing the gene to reflectthe codon use bias of a Pseudomonas species such as P. fluorescens. Thegene(s) that result will have been constructed within or will beinserted into one or more vector, which will then be transformed intothe expression host cell Nucleic acid or a polynucleotide said to beprovided in an “expressible form” means nucleic acid or a polynucleotidethat contains at least one gene that can be expressed by the selectedbacterial expression host cell.

Regulatory Elements

The regulatory elements used herein can be operably attached to thetarget recombinant mammalian protein encoding gene. The coding sequenceof the protein-encoding gene used herein can contain, in addition to themature polypeptide coding sequence and transcription-regulatoryelements, further encoding elements, e.g., one or more of codingsequences for peptide tags, pre-peptides, pro-peptides,pre-pro-peptides, or other commonly utilized encoding elements known inthe art, excluding secretion signal peptides functional in the selectedexpression host cell.

The term “operably attached,” as used herein, refers to anyconfiguration in which the transcriptional and any translationalregulatory elements are covalently attached to the coding sequence insuch disposition(s), relative to the coding sequence, that theregulatory elements can direct the expression of the coding sequence. Inone embodiment, the regulatory elements will be part of a whole genebefore undergoing transformation into a host cell; however, in otherembodiments the regulatory elements are part of another gene, which canbe part of the host genome or can be part of a genome of anotherorganism, or can be derived therefrom.

Promoters and Accessory Elements

The promoters used in accordance with the present invention may beconstitutive promoters or regulated promoters. Common examples of usefulregulated promoters include those of the family derived from the lacpromoter (i.e. the lacZ promoter), especially the tac and trc promotersdescribed in U.S. Pat. No. 4,551,433 to DeBoer, as well as Ptac16,Ptac17, Ptacll, PlacUV5, and the T7lac promoter.

Common examples of non-lac-type promoters useful in expression systemsaccording to the present invention include, e.g., those listed in Table4.

TABLE 4 Examples of non-lac Promoters Promoter Inducer λP_(R) Hightemperature λP_(L) High temperature Pm Alkyl- or halo-benzoates PuAlkyl- or halo-toluenes Psal Salicylates

See, e.g.: J. Sanchez-Romero & V. De Lorenzo (1999) Manual of IndustrialMicrobiology and Biotechnology (A. Demain & J. Davies, eds.) pp.460-74(ASM Press, Washington, D.C.); H. Schweizer (2001) Current Opinion inBiotechnology 12:439-445; and R. Slater & R. Williams (2000) MolecularBiology and Biotechnology (J. Walker & R. Rapley, eds.) pp.125-54. Apromoter having the nucleotide sequence of a promoter native to theselected bacterial host cell may also be used to control expression ofthe transgene encoding the target polypeptide, e.g, a Pseudomonasanthranilate or benzoate operon promoter (Pant, Pben). Tandem promotersmay also be used in which more than one promoter is covalently attachedto another, whether the same or different in sequence, e.g., a Pant-Pbentandem promoter (interpromoter hybrid) or a Plac-Plac tandem promoter.

Regulated promoters utilize promoter regulatory proteins in order tocontrol transcription of the gene of which the promoter is a part. Wherea regulated promoter is used herein, a corresponding promoter regulatoryprotein will also be part of an expression system according to thepresent invention. Examples of promoter regulatory proteins include:activator proteins, e.g., E. coli catabolite activator protein, MalTprotein; AraC family transcriptional activators; repressor proteins,e.g., E. coli Lad proteins; and dual-fuction regulatory proteins, e.g.,E. coli NagC protein. Manyregulated-promoter/promoter-regulatory-protein pairs are known in theart.

Promoter regulatory proteins interact with an effector compound, i.e. acompound that reversibly or irreversibly associates with the regulatoryprotein so as to enable the protein to either release or bind to atleast one DNA transcription regulatory region of the gene that is underthe control of the promoter, thereby permitting or blocking the actionof a transcriptase enzyme in initiating transcription of the gene.Effector compounds are classified as either inducers or co-repressors,and these compounds include native effector compounds and gratuitousinducer compounds. Manyregulated-promoter/promoter-regulatory-protein/effector-compound triosare known in the art. Although an effector compound can be usedthroughout the cell culture or fermentation, in one embodiment in whicha regulated promoter is used, after growth of a desired quantity ordensity of host cell biomass, an appropriate effector compound is addedto the culture in order directly or indirectly result in expression ofthe desired target gene(s).

By way of example, where a lac family promoter is utilized, a lacI genecan also be present in the system. The lacI gene, which is (normally) aconstitutively expressed gene, encodes the Lac repressor protein (LacIprotein) which binds to the lac operator of these promoters. Thus, wherea lac family promoter is utilized, the lacI gene can also be includedand expressed in the expression system. In the case of the lac promoterfamily members, e.g., the tac promoter, the effector compound is aninducer, such as a gratuitous inducer like IPTG(isopropyl-β-D-1-thiogalactopyranoside, also called“isopropylthiogalactoside”).

Other Elements

Other regulatory elements can be included in an expression construct.Such elements include, but are not limited to, for example,transcriptional enhancer sequences, translational enhancer sequences,other promoters, activators, translational start and stop signals,transcription terminators, cistronic regulators, polycistronicregulators, tag sequences, such as nucleotide sequence “tags” and “tag”peptide coding sequences, which facilitates identification, separation,purification, or isolation of an expressed polypeptide.

At a minimum, a protein-encoding gene according to the present inventioncan include, in addition to the mammalian protein coding sequence, thefollowing regulatory elements operably linked thereto: a promoter, aribosome binding site (RBS), a transcription terminator, translationalstart and stop signals. Useful RBSs can be obtained from any of thespecies useful as host cells in expression systems, such as from theselected host cell. Many specific and a variety of consensus RBSs areknown, e.g., those described in and referenced by D. Frishman et al.(1999) Gene 234(2):257-65; and B. E. Suzek et al. (2001) Bioinformatics17(12):1123-30. In addition, either native or synthetic RBSs may beused, e.g., those described in: EP 0207459 (synthetic RBSs); O. Ikehataet al. (1989) Eur. J. Biochem. 181(3):563-70 (native RBS sequence ofAAGGAAG). Further examples of methods, vectors, and translation andtranscription elements, and other elements useful in the presentinvention are described in, e.g.: U.S. Pat. Nos. 5,055,294 and 5,128,130to Gilroy et al.; 5,281,532 to Rammler et al.; 4,695,455 and 4,861,595to Barnes et al.; 4,755,465 to Gray et al.; and 5,169,760 to Wilcox.

Vectors

Transcription of the DNA encoding the enzymes of the present inventionby Pseudomonas is increased by inserting an enhancer sequence into thevector or plasmid. Typical enhancers are cis-acting elements of DNA,usually about from 10 to 300 bp in size that act on the promoter toincrease its transcription. Examples include various Pseudomonasenhancers, as described elsewhere herein.

Generally, the recombinant expression vectors will include origins ofreplication and selectable markers permitting transformation of thePseudomonas host cell, e.g., the antibiotic-free resistance genes of P.fluorescens, and a promoter derived from a highly-expressed gene todirect transcription of a downstream structural sequence. Such promoterscan be derived from operons encoding the enzymes such as3-phosphoglycerate kinase (PGK), acid phosphatase, or heat shockproteins, among others. The heterologous structural sequence isassembled in appropriate phase with translation initiation andtermination sequences, and, in one embodiment, a leader sequence capableof directing secretion of the translated enzyme. Optionally, theheterologous sequence can encode a fusion enzyme including an N-terminalidentification peptide imparting desired characteristics, e.g.,stabilization or simplified purification of expressed recombinantproduct.

Useful expression vectors for use with P. fluorescens in expressingenzymes are constructed by inserting a structural DNA sequence encodinga desired protein together with suitable translation initiation andtermination signals in operable reading phase with a functionalpromoter. The vector will comprise one or more phenotypic selectablemarkers and an origin of replication to ensure maintenance of the vectorand to, if desirable, provide amplification within the host.

Vectors are known in the art as useful for expressing recombinantproteins in host cells, and any of these may be used for expressing thegenes according to the present invention. Such vectors include, e.g.,plasmids, cosmids, and phage expression vectors. Examples of usefulplasmid vectors include, but are not limited to, the expression plasmidspBBR1MCS, pDSK519, pKT240, pML122, pPS10, RK2, RK6, pRO1600, andRSF1010. Other examples of such useful vectors include those describedby, e.g.: N. Hayase (1994) Appl. Envir. Microbiol. 60(9):3336-42; A. A.Lushnikov et al. (1985) Basic Life Sci. 30:657-62; S. Graupner & W.Wackernagel (2000) Biomolec. Eng. 17(1):11-16.; H. P. Schweizer (2001)Curr. Opin. Biotech. 12(5):439-45; M. Bagdasarian & K. N. Timmis (1982)Curr. Topics Microbiol. Immunol. 96:47-67; T. Ishii et al. (1994) FEMSMicrobiol. Lett. 116(3):307-13; I. N. Olekhnovich & Y. K. Fomichev(1994) Gene 140(1):63-65; M. Tsuda & T. Nakazawa (1993) Gene136(1-2):257-62; C. Nieto et al. (1990) Gene 87(1):145-49; J. D. Jones &N. Gutterson (1987) Gene 61(3):299-306; M. Bagdasarian et al. (1981)Gene 16(13):237-47; H. P. Schweizer et al. (2001) Genet. Eng. (NY)23:69-81; P. Mukhopadhyay et al. (1990) J. Bact. 172(1):477-80; D. O.Wood et al. (1981) J. Bact. 145(3):1448-51; and R. Holtwick et al.(2001) Microbiology 147(Pt 2):337-44.

Further examples of expression vectors that can be useful in Pseudomonashost cells include those listed in Table 5 as derived from the indicatedreplicons.

TABLE 5 Some Examples of Useful Expression Vectors Replicon Vector(s)pPS10 pCN39, pCN51 RSF1010 pKT261-3 pMMB66EH pEB8 pPLGN1 pMYC1050RK2/RP1 pRK415 pJB653 pRO1600 pUCP pBSP

The expression plasmid, RSF1010, is described, e.g., by F. Heffron etal. (1975) Proc. Nat'l Acad. Sci. USA 72(9):3623-27, and by K. Nagahari& K. Sakaguchi (1978) J. Bact. 133(3):1527-29. Plasmid RSF1010 andderivatives thereof are particularly useful vectors in the presentinvention. Exemplary, useful derivatives of RSF1010, which are known inthe art, include, e.g., pKT212, pKT214, pKT231 and related plasmids, andpMYC1050 and related plasmids (see, e.g., U.S. Pat. Nos. 5,527,883 and5,840,554 to Thompson et al.), such as, e.g., pMYC1803. Plasmid pMYC1803is derived from the RSF1010-based plasmid pTJS260 (see U.S. Pat. No.5,169,760 to Wilcox), which carries a regulated tetracycline resistancemarker and the replication and mobilization loci from the RSF1010plasmid. Other exemplary useful vectors include those described in U.S.Pat. No. 4,680,264 to Puhler et al.

In a one embodiment, an expression plasmid is used as the expressionvector. In another embodiment, RSF1010 or a derivative thereof is usedas the expression vector. In still another embodiment, pMYC1050 or aderivative thereof, or pMYC1803 or a derivative thereof, is used as theexpression vector.

The plasmid can be maintained in the host cell by use of a selectionmarker gene, also present in the plasmid. This may be an antibioticresistance gene(s), in which case the corresponding antibiotic(s) willbe added to the fermentation medium, or any other type of selectionmarker gene known as useful in the art, e.g., a prototrophy-restoringgene in which case the plasmid will be used in a host cell that isauxotrophic for the corresponding trait, e.g., a biocatalytic trait suchas an amino acid biosynthesis or a nucleotide biosynthesis trait or acarbon source utilization trait.

Extensive sequence information required for molecular genetics andgenetic engineering techniques is widely publicly available. Access tocomplete nucleotide sequences of mammalian, as well as human, genes,cDNA sequences, amino acid sequences, and genomes can be obtained fromGenBank at the URL address http://www.ncbi.nlm.nih.gov/Entrez.Additional information can also be obtained from GeneCards, anelectronic encyclopedia integrating information about genes and theirproducts and biomedical applications from the Weizmann Institute ofScience Genome and Bioinformatics(http://bioinformatics.weizmann.ac.il/cards/), nucleotide sequenceinformation can be also obtained from the EMBL Nucleotide SequenceDatabase (http://www.ebi.ac.uk/embl/) or the DNA Databank or Japan(DDBJ, http://www.ddbj.nig.ac.jp/; additional sites for information onamino acid sequences include Georgetown's protein information resourcewebsite (http://www-nbrf.georgetown.edu/pir/) and Swiss-Prot(http://au.expasy.org/sprot/sprot-top.html).

Transformation

Transformation of the Pseudomonas host cells with the vector(s) may beperformed using any transformation methodology known in the art, and thebacterial host cells may be transformed as intact cells or asprotoplasts (i.e. including cytoplasts). Exemplary transformationmethodologies include poration methodologies, e.g., electroporation,protoplast fusion, bacterial conjugation, and divalent cation treatment,e.g., calcium chloride treatment or CaCl/Mg²⁺ treatment, or other wellknown methods in the art. See, e.g., Morrison (1977) J Bact.132:349-351; Clark-Curtiss & Curtiss (1983) Methods in Enzymology101:347-362, Sambrook et al. (1989) Molecular Cloning, A LaboratoryManual (2nd ed.); Kriegler (1990) Gene Transfer and Expression: ALaboratory Manual; and Ausubel et al., eds. (1994) Current Protocols inMolecular Biology.

Pseudomonas Organisms

While the primary invention herein is the use of Pseudomonasfluorescens, other Pseudomonas and closely related bacterial organismscan be useful. Pseudomonas and closely related bacteria are generallypart of the group defined as “Gram(−) Proteobacteria Subgroup 1” or“Gram-Negative Aerobic Rods and Cocci” (Buchanan and Gibbons (eds.)(1974) Bergey's Manual of Determinative Bacteriology, pp. 217-289).

TABLE 6 “Gram-Negative Aerobic Rods and Cocci” (Bergey (1974)) Family I.Pseudomonadaceae Gluconobacter Pseudomonas Xanthomonas Zoogloea FamilyII. Azotobacteraceae Azomonas Azotobacter Beijerinckia Derxia FamilyIII. Rhizobiaceae Agrobacterium Rhizobium Family IV. MethylomonadaceaeMethylococcus Methylomonas Family V. Halobacteriaceae HalobacteriumHalococcus Other Genera Acetobacter Alcaligenes Bordetella BrucellaFrancisella Thermus

“Gram-negative Proteobacteria Subgroup 1” also includes Proteobacteriathat would be classified in this heading according to the criteria usedin the classification. The heading also includes groups that werepreviously classified in this section but are no longer, such as thegenera Acidovorax, Brevundimonas, Burkholderia, Hydrogenophaga,Oceanimonas, Ralstonia, and Stenotrophomonas, the genus Sphingomonas(and the genus Blastomonas, derived therefrom), which was created byregrouping organisms belonging to (and previously called species of) thegenus Xanthomonas, the genus Acidomonas, which was created by regroupingorganisms belonging to the genus Acetobacter as defined in Bergey(1974). In addition hosts can include cells from the genus Pseudomonas,Pseudomonas enalia (ATCC 14393), Pseudomonas nigrifaciens (ATCC 19375),and Pseudomonas putrefaciens (ATCC 8071), which have been reclassifiedrespectively as Alteromonas haloplanktis, Alteromonas nigrifaciens, andAlteromonas putrefaciens. Similarly, e.g., Pseudomonas acidovorans (ATCC15668) and Pseudomonas testosteroni (ATCC 11996) have since beenreclassified as Comamonas acidovorans and Comamonas testosteroni,respectively; and Pseudomonas nigrifaciens (ATCC 19375) and Pseudomonaspiscicida (ATCC 15057) have been reclassified respectively asPseudoalteromonas nigrifaciens and Pseudoalteromonas piscicida.“Gram-negative Proteobacteria Subgroup 1” also includes Proteobacteriaclassified as belonging to any of the families: Pseudomonadaceae,Azotobacteraceae (now often called by the synonym, the “Azotobactergroup” of Pseudomonadaceae), Rhizobiaceae, and Methylomonadaceae (nowoften called by the synonym, “Methylococcaceae”). Consequently, inaddition to those genera otherwise described herein, furtherProteobacterial genera falling within “Gram-negative ProteobacteriaSubgroup 1” include: 1) Azotobacter group bacteria of the genusAzorhizophilus; 2) Pseudomonadaceae family bacteria of the generaCellvibrio, Oligella, and Teredinibacter; 3) Rhizobiaceae familybacteria of the genera Chelatobacter, Ensifer, Liberibacter (also called“Candidatus Liberibacter”), and Sinorhizobium; and 4) Methylococcaceaefamily bacteria of the genera Methylobacter, Methylocaldum,Methylomicrobium, Methylosarcina, and Methylosphaera .

In another embodiment, the host cell is selected from “Gram-negativeProteobacteria Subgroup 2.” “Gram-negative Proteobacteria Subgroup 2” isdefined as the group of Proteobacteria of the following genera (with thetotal numbers of catalog-listed, publicly-available, deposited strainsthereof indicated in parenthesis, all deposited at ATCC, except asotherwise indicated): Acidomonas (2); Acetobacter (93); Gluconobacter(37); Brevundimonas (23); Beijerinckia (13); Derxia (2); Brucella (4);Agrobacterium (79); Chelatobacter (2); Ensifer (3); Rhizobium (144);Sinorhizobium (24); Blastomonas (1); Sphingomonas (27); Alcaligenes(88); Bordetella (43); Burkholderia (73); Ralstonia (33); Acidovorax(20); Hydrogenophaga (9); Zoogloea (9); Methylobacter (2); Methylocaldum(1 at NCIMB); Methylococcus (2); Methylomicrobium (2); Methylomonas (9);Methylosarcina (1); Methylosphaera; Azomonas (9); Azorhizophilus (5);Azotobacter (64); Cellvibrio (3); Oligella (5); Pseudomonas (1139);Francisella (4); Xanthomonas (229); Stenotrophomonas (50); andOceanimonas (4).

Exemplary host cell species of “Gram-negative Proteobacteria Subgroup 2”include, but are not limited to the following bacteria (with the ATCC orother deposit numbers of exemplary strain(s) thereof shown inparenthesis): Acidomonas methanolica (ATCC 43581); Acetobacter aceti(ATCC 15973); Gluconobacter oxydans (ATCC 19357); Brevundimonas diminuta(ATCC 11568); Beijerinckia indica (ATCC 9039 and ATCC 19361); Derxiagummosa (ATCC 15994); Brucella melitensis (ATCC 23456), Brucella abortus(ATCC 23448); Agrobacterium tumefaciens (ATCC 23308), Agrobacteriumradiobacter (ATCC 19358), Agrobacterium rhizogenes (ATCC 11325);Chelatobacter heintzii (ATCC 29600); Ensifer adhaerens (ATCC 33212);Rhizobium leguminosarum (ATCC 10004); Sinorhizobium fredii (ATCC 35423);Blastomonas natatoria (ATCC 35951); Sphingomonas paucimobilis (ATCC29837); Alcaligenes faecalis (ATCC 8750); Bordetella pertussis (ATCC9797); Burkholderia cepacia (ATCC 25416); Ralstonia pickettii (ATCC27511); Acidovorax facilis (ATCC 11228); Hydrogenophaga flava (ATCC33667); Zoogloea ramigera (ATCC 19544); Methylobacter luteus (ATCC49878); Methylocaldum gracile (NCIMB 11912); Methylococcus capsulatus(ATCC 19069); Methylomicrobium agile (ATCC 35068); Methylomonasmethanica (ATCC 35067); Methylosarcina fibrata (ATCC 700909);Methylosphaera hansonii (ACAM 549); Azomonas agilis (ATCC 7494);Azorhizophilus paspali (ATCC 23833); Azotobacter chroococcum (ATCC9043); Cellvibrio mixtus (UQM 2601); Oligella urethralis (ATCC 17960);Pseudomonas aeruginosa (ATCC 10145), Pseudomonas fluorescens (ATCC35858); Francisella tularensis (ATCC 6223); Stenotrophomonas maltophilia(ATCC 13637); Xanthomonas campestris (ATCC 33913); and Oceanimonasdoudoroffii (ATCC 27123).

In another embodiment, the host cell is selected from “Gram-negativeProteobacteria Subgroup 3.” “Gram-negative Proteobacteria Subgroup 3” isdefined as the group of Proteobacteria of the following genera:Brevundimonas; Agrobacterium; Rhizobium; Sinorhizobium; Blastomonas;Sphingomonas; Alcaligenes; Burkholderia; Ralstonia; Acidovorax;Hydrogenophaga; Methylobacter; Methylocaldum; Methylococcus;Methylomicrobium; Methylomonas; Methylosarcina; Methylosphaera;Azomonas; Azorhizophilus; Azotobacter; Cellvibrio; Oligella;Pseudomonas; Teredinibacter; Francisella; Stenotrophomonas; Xanthomonas;and Oceanimonas.

In another embodiment, the host cell is selected from “Gram-negativeProteobacteria Subgroup 4.” “Gram-negative Proteobacteria Subgroup 4” isdefined as the group of Proteobacteria of the following genera:Brevundimonas; Blastomonas; Sphingomonas; Burkholderia; Ralstonia;Acidovorax; Hydrogenophaga; Methylobacter; Methylocaldum; Methylococcus;Methylomicrobium; Methylomonas; Methylosarcina; Methylosphaera;Azomonas; Azorhizophilus; Azotobacter; Cellvibrio; Oligella;Pseudomonas; Teredinibacter; Francisella; Stenotrophomonas; Xanthomonas;and Oceanimonas.

In an embodiment, the host cell is selected from “Gram-negativeProteobacteria Subgroup 5.” “Gram-negative Proteobacteria Subgroup 5” isdefined as the group of Proteobacteria of the following genera:Methylobacter; Methylocaldum; Methylococcus; Methylomicrobium;Methylomonas; Methylosarcina; Methylosphaera; Azomonas; Azorhizophilus;Azotobacter; Cellvibrio; Oligella; Pseudomonas; Teredinibacter;Francisella; Stenotrophomonas; Xanthomonas; and Oceanimonas.

The host cell can be selected from “Gram-negative ProteobacteriaSubgroup 6.” “Gram-negative Proteobacteria Subgroup 6” is defined as thegroup of Proteobacteria of the following genera: Brevundimonas;Blastomonas; Sphingomonas; Burkholderia; Ralstonia; Acidovorax;Hydrogenophaga; Azomonas; Azorhizophilus; Azotobacter; Cellvibrio;Oligella; Pseudomonas; Teredinibacter; Stenotrophomonas; Xanthomonas;and Oceanimonas.

The host cell can be selected from “Gram-negative ProteobacteriaSubgroup 7.” “Gram-negative Proteobacteria Subgroup 7” is defined as thegroup of Proteobacteria of the following genera: Azomonas;Azorhizophilus; Azotobacter; Cellvibrio; Oligella; Pseudomonas;Teredinibacter; Stenotrophomonas; Xanthomonas; and Oceanimonas.

The host cell can be selected from “Gram-negative ProteobacteriaSubgroup 8.” “Gram-negative Proteobacteria Subgroup 8” is defined as thegroup of Proteobacteria of the following genera: Brevundimonas;Blastomonas; Sphingomonas; Burkholderia; Ralstonia; Acidovorax;Hydrogenophaga; Pseudomonas; Stenotrophomonas; Xanthomonas; andOceanimonas.

The host cell can be selected from “Gram-negative ProteobacteriaSubgroup 9.” “Gram-negative Proteobacteria Subgroup 9” is defined as thegroup of Proteobacteria of the following genera: Brevundimonas;Burkholderia; Ralstonia; Acidovorax; Hydrogenophaga; Pseudomonas;Stenotrophomonas; and Oceanimonas.

The host cell can be selected from “Gram-negative ProteobacteriaSubgroup 10.” “Gram-negative Proteobacteria Subgroup 10” is defined asthe group of Proteobacteria of the following genera: Burkholderia;Ralstonia; Pseudomonas; Stenotrophomonas; and Xanthomonas.

The host cell can be selected from “Gram-negative ProteobacteriaSubgroup 11.” “Gram-negative Proteobacteria Subgroup 11” is defined asthe group of Proteobacteria of the genera: Pseudomonas;Stenotrophomonas; and Xanthomonas. The host cell can be selected from“Gram-negative Proteobacteria Subgroup 12.” “Gram-negativeProteobacteria Subgroup 12” is defined as the group of Proteobacteria ofthe following genera: Burkholderia; Ralstonia; Pseudomonas. The hostcell can be selected from “Gram-negative Proteobacteria Subgroup 13.”“Gram-negative Proteobacteria Subgroup 13” is defined as the group ofProteobacteria of the following genera: Burkholderia; Ralstonia;Pseudomonas; and Xanthomonas. The host cell can be selected from“Gram-negative Proteobacteria Subgroup 14.” “Gram-negativeProteobacteria Subgroup 14” is defined as the group of Proteobacteria ofthe following genera: Pseudomonas and Xanthomonas. The host cell can beselected from “Gram-negative Proteobacteria Subgroup 15.” “Gram-negativeProteobacteria Subgroup 15” is defined as the group of Proteobacteria ofthe genus Pseudomonas.

The host cell can be selected from “Gram-negative ProteobacteriaSubgroup 16.” “Gram-negative Proteobacteria Subgroup 16” is defined asthe group of Proteobacteria of the following Pseudomonas species (withthe ATCC or other deposit numbers of exemplary strain(s) shown inparenthesis): P. abietaniphila (ATCC 700689); P. aeruginosa (ATCC10145); P. alcaligenes (ATCC 14909); P. anguilliseptica (ATCC 33660); P.citronellolis (ATCC 13674); P. flavescens (ATCC 51555); P. mendocina(ATCC 25411); P. nitroreducens (ATCC 33634); P. oleovorans (ATCC 8062);P. pseudoalcaligenes (ATCC 17440); P. resinovorans (ATCC 14235); P.straminea (ATCC 33636); P. agarici (ATCC 25941); P. alcaliphila; P.alginovora; P. andersonii; P. asplenii (ATCC 23835); P. azelaica (ATCC27162); P. beijerinckii (ATCC 19372); P. borealis; P. boreopolis (ATCC33662); P. brassicacearum; P. butanovora (ATCC 43655); P. cellulosa(ATCC 55703); P. aurantiaca (ATCC 33663); P. chlororaphis (ATCC 9446,ATCC 13985, ATCC 17418, ATCC 17461); P. fragi (ATCC 4973); P. lundensis(ATCC 49968); P. taetrolens (ATCC 4683); P. cissicola (ATCC 33616); P.coronafaciens; P. diterpeniphila; P. elongata (ATCC 10144); P. flectens(ATCC 12775); P. azotoformans; P. brenneri; P. cedrella; P. corrugata(ATCC 29736); P. extremorientalis; P. fluorescens (ATCC 35858); P.gessardii; P. libanensis; P. mandelii (ATCC 700871); P. marginalis (ATCC10844); P. migulae; P. mucidolens (ATCC 4685); P. orientalis; P.rhodesiae; P. synxantha (ATCC 9890); P. tolaasii (ATCC 33618); P.veronii (ATCC 700474); P. frederiksbergensis; P. geniculata (ATCC19374); P. gingeri; P. graminis; P. grimontii; P. halodenitrificans; P.halophila; P. hibiscicola (ATCC 19867); P. huttiensis (ATCC 14670); P.hydrogenovora; P. jessenii (ATCC 700870); P. kilonensis; P. lanceolata(ATCC 14669); P. lini; P. marginata (ATCC 25417); P. mephitica (ATCC33665); P. denitrificans (ATCC 19244); P. pertucinogena (ATCC 190); P.pictorum (ATCC 23328); P. psychrophila; P. fulva (ATCC 31418); P.monteilii (ATCC 700476); P. mosselii; P. oryzihabitans (ATCC 43272); P.plecoglossicida (ATCC 700383); P. putida (ATCC 12633); P. reactans; P.spinosa (ATCC 14606); P. balearica; P. luteola (ATCC 43273); P. stutzeri(ATCC 17588); P. amygdali (ATCC 33614); P. avellanae (ATCC 700331); P.caricapapayae (ATCC 33615); P. cichorii (ATCC 10857); P. ficuserectae(ATCC 35104); P. fuscovaginae; P. meliae (ATCC 33050); P. syringae (ATCC19310); P. viridiflava (ATCC 13223); P. thermocarboxydovorans (ATCC35961); P. thermotolerans; P. thivervalensis; P. vancouverensis (ATCC700688); P. wisconsinensis; and P. xiamenensis.

The host cell can be selected from “Gram-negative ProteobacteriaSubgroup 17.” “Gram-negative Proteobacteria Subgroup 17” is defined asthe group of Proteobacteria known in the art as the “fluorescentPseudomonads” including those belonging, e.g., to the followingPseudomonas species: P. azotoformans; P. brenneri; P. cedrella; P.corrugata; P. extremorientalis; Pseudomonas fluorescens; P. gessardii;P. libanensis; Pseudomonas mandelii; P. marginalis; P. migulae; P.mucidolens; P. orientalis; P. rhodesiae; P. synxantha; P. tolaasii; andP. veronii.

The host cell can be selected from “Gram(−) Proteobacteria Subgroup 18.”“Gram(−) Proteobacteria Subgroup 18” is defined as the group of allsubspecies, varieties, strains, and other sub-special units of thespecies P. fluorescens, including those belonging, e.g., to thefollowing (with the ATCC or other deposit numbers of exemplary strain(s)shown in parenthesis): P. fluorescens biotype A, also called biovar 1 orbiovar I (ATCC 13525); P. fluorescens biotype B, also called biovar 2 orbiovar II (ATCC 17816); P. fluorescens biotype C, also called biovar 3or biovar III (ATCC 17400); P. fluorescens biotype F, also called biovar4 or biovar IV (ATCC 12983); P. fluorescens biotype G, also calledbiovar 5 or biovar V (ATCC 17518); P. fluorescens biovar VI; P.fluorescens Pf0-1; P. fluorescens Pf-5 (ATCC BAA-477); P. fluorescensSBW25; and P. fluorescens subsp. cellulosa (NCIMB 10462).

The host cell can be selected from “Gram(−) Proteobacteria Subgroup 19.”“Gram(−) Proteobacteria Subgroup 19” is defined as the group of allstrains of P. fluorescens biotype A. A particular strain of this biotypeis P. fluorescens strain MB101 (see U.S. Pat. No. 5,169,760 to Wilcox),and derivatives thereof. An example of a derivative thereof is P.fluorescens strain MB214, constructed by inserting into the MB101chromosomal asd (aspartate dehydrogenase gene) locus, a native E. coliPlacI-lacI-lacZYA construct (i.e. in which PlacZ was deleted).

In one embodiment, the host cell is any of the Proteobacteria of theorder Pseudomonadales. In a particular embodiment, the host cell is anyof the Proteobacteria of the family Pseudomonadaceae.

Additional P. fluorescens strains that can be used in the presentinvention include P. fluorescens Migula and P. fluorescens Loitokitok,having the following ATCC designations: (NCIB 8286); NRRL B-1244; NCIB8865 strain CO1; NCIB 8866 strain CO2; 1291 (ATCC 17458; IFO 15837; NCIB8917; LA; NRRL B-1864; pyrrolidine; PW2 (ICMP 3966; NCPPB 967; NRRLB-899); 13475; NCTC 10038; NRRL B-1603 (6; IFO 15840); 52-1C; CCEB 488-A(BU 140); CCEB 553 (IEM 15/47); IAM 1008 (AHH-27); IAM 1055 (AHH-23); 1(IFO 15842); 12 (ATCC 25323; NIH 11; den Dooren de Jong 216); 18 (IFO15833; WRRL P-7); 93 (TR-10); 108 (52-22; IFO 15832); 143 (IFO 15836;PL); 149 (2-40-40; IFO 15838); 182 (IFO 3081; PJ 73); 184 (IFO 15830);185 (W2 L-1); 186 (IFO 15829; PJ 79); 187 (NCPPB 263); 188 (NCPPB 316);189 (PJ227; 1208); 191 (IFO 15834; PJ 236; 22/1); 194 (Klinge R-60; PJ253); 196 (PJ 288); 197 (PJ 290); 198 (PJ 302); 201 (PJ 368); 202 (PJ372); 203 (PJ 376); 204 (IFO 15835; PJ 682); 205 (PJ 686); 206 (PJ 692);207 (PJ 693); 208 (PJ 722); 212 (PJ 832); 215 (PJ 849); 216 (PJ 885);267 (B-9); 271 (B-1612); 401 (C71A; IFO 15831; PJ 187); NRRL B-3178 (4;IFO 15841); KY 8521; 3081; 30-21; (IFO 3081); N; PYR; PW; D946-B83 (BU2183; FERM-P 3328); P-2563 (FERM-P 2894; IFO 13658); IAM-1126 (43F);M-1; A506 (A5-06); A505 (A5-05-1); A526 (A5-26); B69; 72; NRRL B-4290;PMW6 (NCIB 11615); SC 12936; Al (IFO 15839); F 1847 (CDC-EB); F 1848(CDC 93); NCIB 10586; P17; F-12; AmMS 257; PRA25; 6133D02; 6519E01; N1;SC15208; BNL-WVC; NCTC 2583 (NCIB 8194); H13; 1013 (ATCC 11251; CCEB295); IFO 3903; 1062; or Pf-5.

Fermentation

The term “fermentation” includes both embodiments in which literalfermentation is employed and embodiments in which other,non-fermentative culture modes are employed. Fermentation may beperformed at any scale. In one embodiment, the fermentation medium maybe selected from among rich media, minimal media, and mineral saltsmedia; a rich medium may be used, but is typically avoided. In anotherembodiment either a minimal medium or a mineral salts medium isselected. In still another embodiment, a minimal medium is selected.

Mineral salts media consists of mineral salts and a carbon source suchas, e.g., glucose, sucrose, or glyceroL Examples of mineral salts mediainclude, e.g., M9 medium, Pseudomonas medium (ATCC 179), Davis andMingioli medium (see, B D Davis & E S Mingioli (1950) J. Bact.60:17-28). The mineral salts used to make mineral salts media includethose selected from among, e.g., potassium phosphates, ammonium sulfateor chloride, magnesium sulfate or chloride, and trace minerals such ascalcium chloride, borate, and sulfates of iron, copper, manganese, andzinc. Typically, no organic nitrogen source, such as peptone, tryptone,amino acids, or a yeast extract, is included in a mineral salts medium.Instead, an inorganic nitrogen source is used and this may be selectedfrom among, e.g., ammonium salts, aqueous ammonia, and gaseous ammonia.A mineral salts medium will typically contain glucose as the carbonsource. In comparison to mineral salts media, minimal media can alsocontain mineral salts and a carbon source, but can be supplemented with,e.g., low levels of amino acids, vitamins, peptones, or otheringredients, though these are added at very minimal levels.

In one embodiment, media can be prepared using the components listed inTable 7 below. The components can be added in the following order: first(NH₄)HPO₄, KH₂PO₄, and citric acid can be dissolved in approximately 30liters of distilled water; then a solution of trace elements can beadded, followed by the addition of an antifoam agent, such as Ucolub N115. Then, after heat sterilization (such as at approximately 121° C.),sterile solutions of glucose MgSO₄ and thiamine-HCL can be added.Control of pH at approximately 6.8 can be achieved using aqueousammonia. Sterile distilled water can then be added to adjust the initialvolume to 371 minus the glycerol stock (123 mL). The chemicals arecommercially available from various suppliers, such as Merck. This mediacan allow for a high cell density cultivation (HCDC) for growth ofPseudomonas species and related bacteria. The HCDC can start as a batchprocess which is followed by a two-phase fed-batch cultivation. Afterunlimited growth in the batch part, growth can be controlled at areduced specific growth rate over a period of 3 doubling times in whichthe biomass concentration can increased several fold. Further details ofsuch cultivation procedures is described by Riesenberg, D et al. (1991)“High cell density cultivation of Escherichia coli at controlledspecific growth rate” J Biotechnol 20(1):17-27.

TABLE 7 Medium composition Initial concentration Component KH₂PO₄ 13.3 gl⁻¹ (NH₄)₂HPO₄ 4.0 g l⁻¹ Citric acid 1.7 g l⁻¹ MgSO₄—7H₂O 1.2 g l⁻¹Trace metal solution 10 ml l⁻¹ Thiamin HCl 4.5 mg l⁻¹ Glucose-H₂O 27.3 gl⁻¹ Antifoam Ucolub N115 0.1 ml l⁻¹ Feeding solution MgSO₄—7H₂O 19.7 gl⁻¹ Glucose-H₂O 770 g l⁻¹ NH₃ 23 g Trace metal solution g l⁻¹ Fe(lllcitrate 6 g l⁻¹ MnCl₂—4H₂O 1.5 g l⁻¹ ZmCH₂COOl₂-2H₂O 0.8 g l⁻¹ H₃BO₃ 0.3g l⁻¹ Na₂MoO₄—2H₂O 0.25 g l⁻¹ CoCl₂ 6H₂O 0.25 g l⁻¹ CuCl₂ 2H₂O 0.15 gl⁻¹ ethylene 0.84 g l⁻¹ dinitrilo-tetracetic acid Na₂—2H₂O (TritriplexIII, Merck)

The expression system according to the present invention can be culturedin any fermentation format. For example, batch, fed-batch,semi-continuous, and continuous fermentation modes may be employedherein.

The expression systems according to the present invention are useful fortransgene expression at any scale (i.e. volume) of fermentation. Thus,e.g., microliter-scale, centiliter scale, and deciliter scalefermentation volumes may be used; and 1 Liter scale and largerfermentation volumes can be used. In one embodiment, the fermentationvolume will be at or above 1 Liter. In another embodiment, thefermentation volume will be at or above 5 Liters, 10 Liters, 15 Liters,20 Liters, 25 Liters, 50 Liters, 75 Liters, 100 Liters, 200 Liters, 500Liters, 1,000 Liters, 2,000 Liters, 5,000 Liters, 10,000 Liters, or50,000 Liters.

In the present invention, growth, culturing, and/or fermentation of thetransformed host cells is performed within a temperature rangepermitting survival of the host cells, such as a temperature within therange of about 4° C. to about 55° C., inclusive. In addition, “growth”is used to indicate both biological states of active cell divisionand/or enlargement, as well as biological states in which a non-dividingand/or non-enlarging cell is being metabolically sustained, the latteruse being synonymous with the term “maintenance.”

Cell Density

An additional advantage in using P. fluorescens in expressingrecombinant mammalian proteins includes the capacity of P. fluorescensto be grown in high cell densities compared to E. coli or otherbacterial expression systems. To this end, P. fluorescens expressionssystems according to the present invention can provide a cell density ofabout 20 g/L or more. The P. fluorescens expressions systems accordingto the present invention can likewise provide a cell density of at leastabout 70 g/L, as stated in terms of biomass per volume, the biomassbeing measured as dry cell weight.

In one embodiment, the cell density will be at least 20 g/L. In anotherembodiment, the cell density will be at least 25 g/L, 30 g/L, 35 g/L, 40g/L, 45 g/L, 50 g/L, 60 g/L, 70 g/L, 80 g/L, 90 g/L, 100 g/L, 110 g/L,120 g/L, 130 g/L, 140 g/L, or at least 150 g/L.

In another embodiments, the cell density at induction will be between 20g/L and 150 g/L; 20 g/L and 120 g/L; 20 g/L and 80 g/L; 25 g/L and 80g/L; 30 g/L and 80 g/L; 35 g/L and 80 g/L; 40 g/L and 80 g/L; 45 g/L and80 g/L; 50 g/L and 80 g/L; 50 g/L and 75 g/L; 50 g/L and 70 g/L; 40 g/Land 80 g/L.

Isolation and Purification

The proteins of this invention may be isolated purified to substantialpurity by standard techniques well known in the art, includingincluding, but not limited to, ammonium sulfate or ethanolprecipitation, acid extraction, anion or cation exchange chromatography,phosphocellulose chromatography, hydrophobic interaction chromatography,affinity chromatography, nickel chromatography, hydroxylapatitechromatography, reverse phase chromatography, lectin chromatography,preparative electrophoresis, detergent solubilization, selectiveprecipitation with such substances as column chromatography,immunopurification methods, and others. For example, proteins havingestablished molecular adhesion properties can be reversibly fused aligand. With the appropriate ligand, the protein can be selectivelyadsorbed to a purification column and then freed from the column in arelatively pure form. The fused protein is then removed by enzymaticactivity. In addition, protein can be purified using immunoaffinitycolumns or Ni-NTA columns. General techniques are further described in,for example, R. Scopes (1982) Protein Purification: Principles andPractice, Springer-Verlag: N.Y.; Deutscher (1990) Guide to ProteinPurification, Academic Press; U.S. Pat. No. 4,511,503; S. Roe (2001)Protein Purification Techniques: A Practical Approach, Oxford Press; D.Bollag, et al. (1996) Protein Methods, Wiley-Lisa, Inc.; A K Patra etal. (2000) Protein Expr Purif, 18(2):182-92; and R. Mukhija, et al.(1995) Gene 165(2):303-6. See also, for example, Deutscher (1990) “Guideto Protein Purification,” Methods in Enzymology vol. 182, and othervolumes in this series; Coligan, et al. (1996 and periodic Supplements)Current Protocols in Protein Science, Wiley/Greene, NY; andmanufacturer's literature on use of protein purification products, e.g.,Pharmacia, Piscataway, N.J., or Bio-Rad, Richmond, Calif. Combinationwith recombinant techniques allow fusion to appropriate segments, e.g.,to a FLAG sequence or an equivalent which can be fused via aprotease-removable sequence. See also, for example: Hochuli (1989)Chemische Industrie 12:69-70; Hochuli (1990) “Purification ofRecombinant Proteins with Metal Chelate Absorbent” in Setlow (ed.)Genetic Engineering, Principle and Methods 12:87-98, Plenum Press, NY;and Crowe, et al. (1992) QIAexpress: The High Level Expression & ProteinPurification System QUIAGEN, Inc., Chatsworth, Calif.

Detection of the expressed protein is achieved by methods known in theart and include, for example, radioimmunoassays, Western blottingtechniques, or immunoprecipitation.

The recombinantly produced and expressed enzyme can be recovered andpurified from the recombinant cell cultures by numerous methods, forexample, high performance liquid chromatography (HPLC) can be employedfor final purification steps, as necessary.

Certain proteins expressed in this invention may form insolubleaggregates (“inclusion bodies”). Several protocols are suitable forpurification of proteins from inclusion bodies. For example,purification of inclusion bodies typically involves the extraction,separation and/or purification of inclusion bodies by disruption of thehost cells, e.g., by incubation in a buffer of 50 mM TRIS/HCl pH 7.5, 50mM NaCl, 5 mM MgCl₂, 1 mM DTT, 0.1 mM ATP, and 1 mM PMSF. The cellsuspension is typically lysed using 2-3 passages through a French Press.The cell suspension can also be homogenized using a Polytron (BrinkmanInstruments) or sonicated on ice. Alternate methods of lysing bacteriaare apparent to those of skill in the art (see, e.g., Sambrook, J., E.F. Fritsch and T. Maniatis eds. (1989) “Molecular Cloning: A LaboratoryManual”, 2d ed., Cold Spring Harbor Laboratory Press; Ausubel et al.,eds. (1994) Current Protocols in Molecular Biology).

If necessary, the inclusion bodies can be solubilized, and the lysedcell suspension typically can be centrifuged to remove unwantedinsoluble matter. Proteins that formed the inclusion bodies may berenatured by dilution or dialysis with a compatible buffer. Suitablesolvents include, but are not limited to urea (from about 4 M to about 8M), formamide (at least about 80%, volume/volume basis), and guanidinehydrochloride (from about 4 M to about 8 M). Although guanidinehydrochloride and similar agents are denaturants, this denaturation isnot irreversible and renaturation may occur upon removal (by dialysis,for example) or dilution of the denaturant, allowing re-formation ofimmunologically and/or biologically active protein. Other suitablebuffers are known to those skilled in the art.

Alternatively, it is possible to purify the recombinant proteins fromthe host periplasm. After lysis of the host cell, when the recombinantprotein is exported into the periplasm of the host cell, the periplasmicfraction of the bacteria can be isolated by cold osmotic shock inaddition to other methods known to those skilled in the art. To isolaterecombinant proteins from the periplasm, for example, the bacterialcells can be centrifuged to form a pellet. The pellet can be resuspendedin a buffer containing 20% sucrose. To lyse the cells, the bacteria canbe centrifuged and the pellet can be resuspended in ice-cold 5 mM MgSO₄and kept in an ice bath for approximately 10 minutes. The cellsuspension can be centrifuged and the supernatant decanted and saved.The recombinant proteins present in the supernatant can be separatedfrom the host proteins by standard separation techniques well known tothose of skill in the art.

An initial salt fractionation can separate many of the unwanted hostcell proteins (or proteins derived from the cell culture media) from therecombinant protein of interest. One such example can be ammoniumsulfate. Ammonium sulfate precipitates proteins by effectively reducingthe amount of water in the protein mixture. Proteins then precipitate onthe basis of their solubility. The more hydrophobic a protein is, themore likely it is to precipitate at lower ammonium sulfateconcentrations. A typical protocol includes adding saturated ammoniumsulfate to a protein solution so that the resultant ammonium sulfateconcentration is between 20-30%. This concentration will precipitate themost hydrophobic of proteins. The precipitate is then discarded (unlessthe protein of interest is hydrophobic) and ammonium sulfate is added tothe supernatant to a concentration known to precipitate the protein ofinterest. The precipitate is then solubilized in buffer and the excesssalt removed if necessary, either through dialysis or diafiltration.Other methods that rely on solubility of proteins, such as cold ethanolprecipitation, are well known to those of skill in the art and can beused to fractionate complex protein mixtures.

The molecular weight of a recombinant protein can be used to isolated itfrom proteins of greater and lesser size using ultrafiltration throughmembranes of different pore size (for example, Amicon or Milliporemembranes). As a first step, the protein mixture can be ultrafilteredthrough a membrane with a pore size that has a lower molecular weightcut-off than the molecular weight of the protein of interest. Theretentate of the ultrafiltration can then be ultrafiltered against amembrane with a molecular cut off greater than the molecular weight ofthe protein of interest. The recombinant protein will pass through themembrane into the filtrate. The filtrate can then be chromatographed asdescribed below.

Recombinant protiens can also be separated from other proteins on thebasis of its size, net surface charge, hydrophobicity, and affinity forligands. In addition, antibodies raised against proteins can beconjugated to column matrices and the proteins immunopurified. All ofthese methods are well known in the art. It will be apparent to one ofskill that chromatographic techniques can be performed at any scale andusing equipment from many different manufacturers (e.g., PharmaciaBiotech).

Renaturation and Refolding

Insoluble protein can be renatured or refolded to generate secondary andtieriary protein structure conformation. Protein refolding steps can beused, as necessary, in completing configuration of the recombinantproduct. Refolding and renaturation can be accomplished using an agentthat is known in the art to promote dissociation/association ofproteins. For example, the protein can be incubated with dithiothreitolfollowed by incubation with oxidized glutathione disodium salt followedby incubation with a buffer containing a refolding agent such as urea.

Recombinant protein can also be renatured, for example, by dialyzing itagainst phosphate-buffered saline (PBS) or 50 mM Na-acetate, pH 6 bufferplus 200 mM NaCl. Alternatively, the protein can be refolded whileimmobilized on a column, such as the Ni-NTA column by using a linear6M-1M urea gradient in 500 mM NaCl, 20% glycerol, 20 mM Tris/HCl pH 7.4,containing protease inhibitors. The renaturation can be performed over aperiod of 1.5 hours or more. After renaturation the proteins can beeluted by the addition of 250 mM immidazole. Immidazole can be removedby a final dialyzing step against PBS or 50 mM sodium acetate pH 6buffer plus 200 mM NaCL The purified protein can be stored at 4° C. orfrozen at −80° C.

Other methods include, for example, those that may be described in: M HLee et al. (2002) Protein Expr. Purif. 25(1):166-73; W. K. Cho et al.(2000) J. Biotechnology 77(2-3):169-78; Deutscher (1990) “Guide toProtein Purification,” Methods in Enzymology vol. 182, and other volumesin this series; Coligan, et al. (1996 and periodic Supplements) CurrentProtocols in Protein Science, Wiley/Greene, NY; S. Roe (2001) ProteinPurification Techniques: A Practical Approach, Oxford Press; D. Bollag,et al. (1996) Protein Methods, Wiley-Lisa, Inc.

Active Protein or Peptide Analysis

Typically, an “active” protein includes proteins that have a biologicalfunction or biological effect comparable to the corresponding nativeprotein. In the context of proteins this typically means that apolynucleotide or polypeptide comprises a biological function or effectthat has at least about 20%, about 50%, about 60%, about 70%, about 75%,about 80%, about 85%, about 90%, about 95%, about 98%, or 100%biological function compared to the corresponding native protein usingstandard parameters. The determination of protein activity can beperformed utilizing corresponding standard, targeted comparativebiological assays for particular proteins. One indication that arecombinant protein biological function or effect is that therecombinant polypeptide is immnunologically cross reactive with thenative polypeptide.

Active proteins typically have a specific activity of at least 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, or 95% that of the native mammalianprotein. Further, the substrate specificity (k_(cat)/K_(m)) isoptionally substantially similar to the native mammalian protein.Typically, k_(cat)/K_(m) will be at least 30%, 40%, 50%, 60%, 70%, 80%,or 90% that of the native protein. Methods of assaying and quantifyingmeasures of protein and peptide activity and substrate specificity(k_(cat)/K_(m)), are well known to those of skill in the art.

The activity of a recombinant mammalian protein can be measured by anyprotein specific conventional or standard in vitro or in vivo assayknown in the art. The activity of the Pseudonmonas produced recombinantmammalian protein can be compared with the activity of the correspondingnative mammalian protein to determine whether the recombinant mammalianprotein exhibits substantially similar or equivalent activity to theactivity generally observed in the native protein under the same orsimilar physiological conditions.

The activity of the recombinant protein can be compared with apreviously established native protein standard activity. Alternatively,the activity of the recombinant protein can be determined in asimultaneous, or sunstantially simultaneous, comparative assay with thenative protein. For example, an in vitro assays can be used to determineany detectable interaction between a recombinant protein and a target,e.g. between an expressed enzyme and substrate, between expressedhormone and hormone receptor, between expressed antibody and antigen,etc. Such detection can include the measurement of colorimetric changes,proliferation changes, cell death, cell repelling, changes inradioactivity, changes in solubility, changes in molecular weight asmeasured by gel electrophoresis and/or gel exclusion methods,phosphorylation abilities, antibody specificity assays such as ELISAassays, etc. In addition, in vivo assays include, but are not limitedto, assays to detect physiological effects of the Pseudomonas producedprotein in comparison to physiological effects of the native protein,e.g. weight gain, change in electrolyte balance, change in bloodclotting time, changes in clot dissolution and the induction ofantigenic response. Generally, any in vitro or in vivo assay can be usedto determine the active nature of the Pseudomonas produced recombinantmammalian protein that allows for a comparative analysis to the nativeprotein so long as such activity is assayable. Alternatively, theproteins produced in the present invention can be assayed for theability to stimulate or inhibit interaction between the protein and amolecule that normally interacts with the protein, e.g. a substrate or acomponent of the signal pathway that the native protein normallyinteracts. Such assays can typically include the steps of combining theprotein with a substrate molecule under conditions that allow theprotein to interact with the target molecule, and detect the biochemicalconsequence of the interaction with the protein and the target molecule.

Assays that can be utilized to determine protein activity are described,for example, in: Ralph, P. J., et al. (1984) J. Immunol. 132:1858; Saikiet al. (1981) J. Immunol. 127:1044; Steward, W. E. II (1980) TheInterferon Systems. Springer-Verlag, Vienna and New York; Broxmeyer, H.E., et al. (1982) Blood 60:595; Sambrook, J., E. F. Fritsch and T.Maniatis eds. (1989) “Molecular Cloning: A Laboratory Manual”, 2d ed.,Cold Spring Harbor Laboratory Press; Berger, S. L. and A. R. Kimmel eds.(1987) “Methods in Enzymology: Guide to Molecular Cloning Techniques”,Academic Press; AK Patra et al. (2000) Protein Expr Purif 18(2):182-92;Kodama et al. (1986) J. Biochem. 99:1465-1472; Stewart et al. (1993)Proc. Nat'l Acad. Sci. USA 90:5209-5213; Lombillo et al. (1995) J. CellBiol. 128:107-115; Vale et al. (1985) Cell 42:39-50.

EXAMPLES Bacterial Strains and Growth Conditions.

Unless otherwise specified, all strains used for all Pseudomonasexpression testing were based on P. fluorescens strain MB101. E. colistrains JM109 (Promega), XL2 Blue (Stratagene) or Top 10 (Invitrogen)were used for general cloning. For E. coli expression studies, BL21(DE3)Gold was used. P. fluorescens strains were grown in either LB or minimalsalts medium supplemented with 15 μg/mL tetracycline and 30 μg/mLkanamycin as needed at 30° C. E. coli strains were grown in LBsupplemented with 30 μg/mL kanamycin and/or 15 μg/mL chloramphenicol, or15 μg/mL tetracycline as needed at 37° C. Cells were induced with 0.3 mMIPTG following growth phase.

Protein Activity Detection (ELISA Assay)

Plates were coated by adding 200 μL of the β-galactosidase solution at10 μg/mL in PBS (pH 7.6) to each well of the microtiter plate. Plateswere incubated at room temperature for 16 hrs, then washed 3 times with200 μL PBS +0.1% Tween-20 (PBS-T). Primary antibody was diluted in PBSwith 2% nonfat dry milk (w/v). 200 μL of the diluted antibody was addedto each well and incubated at room temperature for 1 hr. The plates werethen washed 4 times with 200 μL PBS-T. The secondary antibody was alsodiluted in PBS with 2% non fat dry milk (w/v) and to each well, 200 μLwas added and incubated at room temperature for 1.5-2 hours. The plateswere then washed 4 times with PBS-T. A tertiary antibody is used todetect the scFv antibodies: alkaline phosphatase conjugated sheepanti-mouse antibody (Sigma-Aldrich, St. Louis, Mo., USA cat #A5324). Toeach desired well was added 200 μL of diluted antibody solution (orPBS-T) and incubated at room temperature for 1.5 hours. The plates werethen washed 4 times with PBS-T. To each well was added 200 μL of thefreshly prepared Sigma Fast pNPP substrate (Sigma catalogue #R-2770).After 30 minutes, the reaction was stopped by adding 50 μL 3M NaOH toeach well and absorbance was read at 405 nm.

Fermentation

The inoculum for the fermentor culture for P. fluorescens is generatedby inoculating a shake flask containing 600 mL of a chemically definedmedium supplemented with yeast extract and dextrose. Tetracycline istypically added to ensure maintenance of the recombinant plasmid in thestarter culture during its overnight incubation as well as in thefermentor. The shake flask culture is then aseptically transferred to a20 L fermentor containing a chemically defined medium designed tosupport a high biomass, without yeast extract supplementation. Oxygen ismaintained at a positive level in the liquid culture by regulating theair flow into the fermentor and the agitator mixing rate; the pH ismaintained at greater than 6.0 through the addition of aqueous ammonia.The fed-batch high density fermentation process is divided into aninitial growth phase of approximately 24 h and gene expression(induction) phase in which an inducer is added to initiate recombinantgene expression. Glucose, in the form of corn syrup, is fed throughoutthe fermentation process at limiting concentrations. The target celldensity for initiating the induction phase is typically 150 OD units at575 nm. The induction phase of the fermentation is typically allowed togo for approximately 45 to 55 hours. During this phase, samples arewithdrawn from the fermentor for various analyses to determine the levelof target gene expression, cell density, etc.

For each fermentation experiment for E. coli, a frozen glycerol stock isremoved from −80° C. storage, thawed, and diluted before inoculating ashake flask containing 600 mL of LB broth supplemented with kanamycin.The shake flask culture is incubated at 37° C. with shaking at 300 rpmovernight and then aseptically transferred to a 20 L fermentorcontaining complex medium.

Temperature in the fermentor is maintained at 37° C., pH at 7 throughthe addition of aqueous ammonia and phosphoric acid, and dissolvedoxygen at greater than 20%. After a brief initial batch phase, glycerolis fed at rates increased stepwise to maintain excess carbon. When thecell density reaches 24-28 OD units at 600 nm, recombinant expression iseffected by addition of an inducer, such as isopropyl-thiogalactoside(IPTG). The induction phase of the fermentation typically continues forapproximately 3 to 5 hours as the fermentor reached volumetric capacityor as the growth rate began to decrease significantly. During thisphase, samples are withdrawn from the fermentor for various analyses todetermine the level of target gene expression, cell density, etc. Cellfractionation and SDS-PAGE analysis.

Samples are normalized to A575=30, and 1 mL normalized culture ispelleted. Cells are resuspended in 1 mL lysis buffer (50 mM Tris base;200 mM NaCl; 5% v/v glycerol; 20 mM EDTA disodium salt; 0.5% v/v TritonX-100; 1 mM DTT). A protease inhibitor cocktail specific for bacteriallysates (Sigma#P8465) is added to a IX concentration. The resuspendedcells are added to a 2 ml screw cap microfuge tube approximately ¾ fullwith 0.1 mm glass beads and the cells are mechanically lysed using 4, 1minute incubations in a BioSpec bead mill at the highest setting. Cellsare kept on ice between incubations. Approximately 100 μL of lysed cellsolution is removed from beads, transferred into a new tube, andpelleted. The supernatant (soluble fraction) is removed to a new tube.The pellet (insoluble fraction) is resuspended in an equal volume (100μL) of lysis buffer plus protease inhibitor. Five uL of each sample isadded to 5 μL of 2× LDS loading buffer (Invitrogen) and loaded onto a4-12% or 10% Bis-Tris NuPAGE gel (Invitrogen) and run in either 1× MESor 1× MOPS buffer as indicated.

Example 1

Expression of scFV in the Cytoplasm

Single chain antibody fragments (scFV) are finding increased use asdiagnostic and therapeutic agents. These relatively small proteins aremade by fusing together genes coding the variable light and heavy chainsof an immunoglobulin.

Cloning of GalI3 scFv

The GalI3 scFv gene (Genbank accession number AF238290), cloned into thephage display vector pCANTAB6, (see P. Martineau et al. (1998) J. Mol.Biol. 280(1):117-27) was used as template to amplify a 774 base pairproduct, which was subsequently cloned into the pCR2.1 TOPO vector(Invitrogen, Carlsbad, Calif., USA). The scFv gene was excised from theTOPO vector with SpeI and SalI restriction enzymes (New England Biolabs,Beverly, Mass., USA) and cloned into the SpeI and XhoI sites of the P.fluorescens vector pMYC1803, downstream of the Ptac promoter, to producepDOW1117. The resulting plasmids were electroporated into P.fluorescens. The Gall3 gene was cloned into the pET24d+ expressionvector (Novagen, Madison, Wisc., USA), following amplification such thatSalI and NcoI sites flanked the coding sequence. The PCR products weredigested with SalI and NcoI and cloned into the same sites of pET24d+vector downstream of the T7 promoter. The newly formed construct wasthen used to transform XL2 Blue competent cells. Once sequence wasconfirmed, the DNA construct was used to transform BL21(DE3) Gold(Stratagene, San Diego, Calif., USA) for expression.

Expression of a Single Chain Antibody Fragment (scFv) in E. coli and P.fluorescens

scFv molecules were expressed in both E. coli and P. fluorescens, amongthem an scFv with binding activity to the E. coli proteinβ-galactosidase single chain antibody gall3 (P. Martineau et al.,“Expression of an antibody fragment at high levels in the bacterialcytoplasm,” J. Mol. Biol. 280(1):117-27 (1998)). P. fluorescensexpressed about six-fold more protein than E. coli during 20 Lfermentation, with 3.1 g/L yield in P. fluorescens and 0.5 g/L yield inE. coli as determined by SDS-PAGE and densitometry (see Table 8). P.fluorescens expressed about 96% soluble protein, whereas E. coliexpresses only 48% soluble protein.

TABLE 8 Gal13 fermentation summary (*compared to BSA standards) E. coliP. fluorescens Pf/Ec Fermentation Time (hr) 8-9 70 8 Max hGH titre(*g/L) 0.4 (85% cv) 3.1 (24% cv) 8 Dry biomass (g/L) ND (30) 59 (2)hGH/biomass (% w/w) (1) 5 (5)

Material purified from both expression systems was found to be active inan enzyme-linked immunosorbant assay (ELISA) as shown in FIG. 2.Material was also purified from the soluble fraction only of equallysate volumes from lysates of both strains using affinitychromatography. Finally, the overall volumetric recovery for the P.fluorescens process is approximately 20 fold more efficient than for E.coli, 1.34 g/L vs. 0.07 g/L.

Example 2

Expression of human γ-IFN in the CytoplasmCloning of Human gamma-Interferon

Human gamma interferon (hu-γIFN, Genbank accession X13274) was amplifiedfrom a human spleen cDNA library (Invitrogen, Carlsbad, Calif., USA;catalogue #10425-015) such that it lacked the native secretion signal,with the N-terminus of the recombinant γ-IFN beginning asMet-Cys-Tyr-Cys-Gln-Asp-Pro (SEQ ID NO: 28) as described in P W Gray etal. (1982) Nature 298:859-63. The resulting product was cloned into thepCR2.1 TOPO vector and the sequence was confirmed. The hu-γIFN gene wasexcised from the TOPO vector with SpeI and XhoI restriction enzymes andcloned into the same sites of pMYC1803. In a separate reaction, hu-γIFNwas amplified such that AflIII and XhoI sites flanked that codingsequence. The resulting fragment was cloned into the TOPO-TA vector(Invitrogen) and transformed into chemically competent E. coli JM109cells (Promega, Madison, Wisc., USA). The gene was isolated by digestingwith AflIII and XhoI (New England Biolabs), cloned into the NcoI andXhoI sites of pET24d+(Novagen, Madison, Wisc., USA) downstream of the T7promoter, and transformed into JM109. A positive clone was transformedinto E. coli BL21(DE3) cells (Novagen) to test for expression.

Human gamma-Interferon Purification

Frozen cell paste from P. fluorescens cultures was thawed andre-suspended in lysis buffer (50 mM potassium phosphate, pH 7.2containing 50 mM NaCl, 10 mM EDTA (ethylenediaminetetraacetic acid,catalog number BPII8-500, Fisher Scientific, Springfield, N.J., USA), 1mM PMSF (phenylmethylsulfonyl fluoride, catalog number P-7626, Sigma,St. Louis, Mo.), 1 mM dithiothreitol (catalog number D-0632, Sigma), and1 mM benzamidine (catalog number B-6506, Sigma)) at a ratio of about 1gram cell paste per 2 mL lysis buffer. Cells were broken by threepassages through a microfluidizer (model 110Y, MicrofluidicsCorporation, Newton, Mass., USA). Cell debris and unbroken cells wereremoved by centrifugation (for 60 min at 23,708×g and 4° C. using aBeckman Coulter centrifuge; model JA 25.50, Beckman Coulter, Inc.,Fullerton, Calif., USA). The resulting supernatant (cell-free extracts)was clarified by adding 10% w/v diatomaceous earth (Celite product,World Minerals, Inc., Goleta, Calif., USA) and passing the resultthrough a paper filter (Whatman 1, catalog number 1001-150, WhatmanPaper Ltd., Maidstone, Kent, UK)) with vacuum filtration.

Clarified cell extracts were applied to a 3.2 cm×13.5 cm chromatographycolumn of SP-Sepharose FAST FLOW (6% cross-linked agarose bead material;catalog number 17-0709-10, Amersham Biosciences, Piscataway, N.J., USA)equilibrated in buffer A, at a flow rate of 0.5 mL/min. The compositionof Buffer A was: 50 mM HEPES, pH 7.8 (i.e.N-(2-hydroxyethyl)piperazine)N′-(2-ethanesulfonic acid), from FisherScientific, catalog number BP-310-100), 50 mM NaCl, 1 mM EDTA, and 0.02%sodium azide (catalog number 71289, Sigma Chemical Co.). After loading,the column was washed with 3 column volumes (column volume=108 mL)buffer A and 5 column volumes of buffer A containing 0.4 M NaCL Thecolumn was further developed by applying a gradient of 0.4 M to 1 M NaClin the same buffer at a flow rate of 2 mL/min for a total of 7 columnvolumes. Fractions containing pure IFN-γ were then pooled and dialyzedagainst 1×PBS (phosphate-buffered saline, pH 7.2) at 4° C. Protein wasconcentrated by ultrafiltration (using a YM30 ultrafiltration membrane;catalog no. 13722, from Millipore, Bedford, Mass. USA), then frozen inliquid nitrogen and stored at 80° C.

Expression of Human γ-Interferon in E. coli and P. fluorescens

Human γ-interferon is produced commercially by fermentation of E. coliexpressing the γ-IFN gene. The protein is expressed cytoplasmically inan insoluble and inactive form. In order to produce the recombinantpolypeptide as an active pharmaceutical ingredient, the interferon mustbe recovered, solubilized, refolded, and then purified. All these unitoperations add greatly to the cost of goods (COGs) for this protein. Ahuman spleen cDNA library was used as a template to amplify the γIFNcDNA without the native signal sequence and clone into E. coli and P.fluorescens expression vectors. P. fluorescens construct produced ˜4g/Lof γIFN protein during a typical 20 L fermentation reaction. SDS-PAGEand Western analyses of soluble and insoluble fractions show that themajority of the protein (95%) is present in the soluble fraction. FIG. 1shows that hu-γ-IFN purified from the soluble fraction of P. fluorescenssamples displays activity comparable to a commercially availablestandard. FIGS. 5A-5D and Table 9 show a comparison of expression ofγ-IFN between E. coli and P. fluorescens expression systems.

TABLE 9 γ-IFN fermentation summary (*compared to BSA standards) E. coliP. fluorescens Pf/Ec Fermentation Time (hr) 7-9 55 6 Max hGH titre(*g/L) 3.9 4.5 1.5 Dry biomass (g/L) ~22 100 4.5 hGH/biomass (% w/w)~17.7 4.5 0.25Assay of Human gamma Interferon Activity

Cell lines and media: Hela cells (catalogue no. CCL-2) andencephalomyocarditis virus (ECMV, catalogue no. VR-129B) were obtainedfrom the American Type Culture Collection (Manassas, Va.). HeLa cellswere maintained in Eagles Modified Essential Medium (Cellgro EMEM,Mediatech, Herdon, Va., USA) with 10% fetal bovine serum (Gibco,Invitrogen, Carlsbad, Calif., USA) at 37° C./5% CO₂.

The activity of purified hu-γIFN was assayed using a viral inhibitionassay as previously described (J A Lewis (1987) in Lymphokines andInterferons: A Practical Approach M J Clemens et al. (eds.) (IRL PressLtd, Oxford, England). Briefly, HeLa cells were seeded in a 96-wellmicrotiter plate at 3×10⁴ per well. After 24 hours, purified hu-γIFNisolated from P. fluorescens, or E. coli recombinant hu-γIFN (from R&DSystems, Minneapolis, Minn., USA), was added to triplicate wells at 0,0.01, or 0.05 ng per well. After preincubating the cells with hu-γIFNfor 24 hours, ECMV was added at varying dilutions to sets of triplicatewells. The cells were incubated for 5 days, after which cell viabilitywas measured using a cell proliferation ELISA that monitors5-bromo-2′-deoxyuridine incorporation (catalogue no. 1647229, RocheMolecular Biochemicals, Indianapolis, Ind., USA). Results are expressedas absorbance units, with greater absorbance resulting from thepresences of a greater number of actively dividing (live) cells.

Example 3 Expression of hGH in the Cytoplasm

Primers were designed to amplify human growth hormone (hGH) from humancDNA libraries. For this study, hGH was amplified using AmpliTaqpolymerase (Perkin Elmer) according to the manufacturer's protocol,using the above plasmid as template and primers ELVIrev and hgh-sig,with a PCR cycling profile of 95° C. 2 minutes (95° C. 60 seconds 42° C.120 seconds 72° C. 3 minutes) 25×. The resulting product was purifiedusing Wizard PCR DNA purification kit (Promega), digested with SpeI andXhoI restriction enzymes (New England Biolabs) and cloned into the samesites of pMYC1803 (see FIG. 3B). A mutation found in the amplified hGHwas corrected by using the hgh-sigcorr primer with ELVIrev and repeatingthe PCR and cloning procedures.

Primers used to clone hGH.

hGH-sig AGAGAACTAGTAAAAAGGAGAAATCCATGTTCCCAACCATTCCCTTATC (SEQ ID NO: 4) HGH- AGAGAACTAGTAAAAAGGAGAAATCCATGTTCCCAACCATsigcorr TCCCTTATCCAGGCCTTTTGAC (SEQ ID NO: 5) ELVIforAGAGAACTAGTAAAAAGGAGAAATCCATGGCTACAGGCTC CCGGACGTCC (SEQ ID NO: 6)ELVIrev AGAGACTCGAGTCATTAGAAGCCACAGCTGCCCTCCAC (SEQ ID NO: 7)

Purification of hGH

Following 20 L fermentation, hGH was purified from the insolublefraction of E. coli and P. fluorescens cells, with the exception thatduring DEAE FF elution a gradient from 0 to 0.5 M NaC1 was used in placeof a 0.25 M NaCl step.

Expression of Human Growth Hormone in E. coli vs. P. fluorescens.

The cDNA encoding human growth hormone was amplified from a humanpituitary cDNA library. The native secretion signal sequence wasremoved, and an N-terminal methionine was engineered into the constructsfor microbial expression. For E. coli expression, the pET25 vectorcontaining the hGH gene was transformed into BL21(DE3), which containsan integrated T7 polymerase gene necessary for hGH transcription. P.fluorescens expression studies were carried out in the MB214 strain,which contains an integrated lad gene to control expression from thePtac promoter. Both expression systems were evaluated at the 20 Lfermentation scale. As shown in Table 10, P. fluorescens (Pf)outperformed E. coli (EC) in the amount of protein produced per gram ofdry biomass (1.6× as much).

TABLE 10 hGH fermentation summary (*compared to BSA standards) E. coliP. fluorescens Pf/Ec Fermentation Time (hr) 7-9 55 6 Max hGH titre(*g/L) 2.6 (23% cv) 7.3 (5% cv) 3 Dry biomass (g/L) 37 66 2 hGH/biomass(% w/w) 7 11 1.6

Cell fractionation and SDS-PAGE analysis show that hGH is found in theinsoluble fraction in both expression systems (FIGS. 4A and 4B).Surprisingly, approximately 7× more hGH monomer was purified from P.fluorescens, compared to E.coli, despite a difference of only 1.6× inprotein production per gram of dry biomass.

TABLE 11 Comparison of hGH purification from E. coli and P. fluorescensWet Biomass Purified Monomer Purified Dimer (wet g) (mg) (mg) 1.62-1.75L portion of P. fluorescens containing rh-GH Soluble minimal NA NA LipidExtract None detected — — Lipid Insoluble 62.2-63.1 1483 346 1.5-1.6 Lportion of E. coli containing rh-GH Soluble minimal NA NA Lipid ExtractNone detected — — Lipid Insoluble 35.0  200 333

Example 4 Expression of Proteins in the Periplasm Characterization ofSecretion Signal Peptides

Pseudomonas fluorescens secretion signal peptides were discovered byformation and expression of alkaline phosphatase (phoA) codingsequence-genomic DNA fusions and are described in more detail in U.S.application Ser. No. 10/996,007, filed Nov. 22, 2004. Six of theexpressed fusions were further characterized as follows.

The cleavage site for the signal sequences for the secreted genesidentified as phoA fusions was deduced by comparison to homolgousproteins from other Pseudomonads, by the SPScan program (Menne et al,2000). The cleavage site of the putative lipoprotein was deduced bycomparison to signal peptidase II motifs; signal peptidase IIspecifically cleaves the signal sequences of lipoproteins. All six ofthe signal peptides were analyzed using SignalP (a software program foranalysis of putative signal peptides; available from the Center forBiological Sequence Analysis of the Technical University of Denmark, athttp://www.cbs.dtu.dklservices/SignalP/.) Also see, Nielson et al.(1997) Protein Engineering 10:1-6. In some cases, a supplementary sourcewas used to further characterize the identity of the signal peptide.This information is present in Table 12.

TABLE 12 Identities of Secretion Signal Peptides IdentityPutative Amino Acid Sequence Putative porin E1Lys Lys Ser Thr Leu Ala Val Ala Val Thr Leu Gly Ala Ile Ala Glnprecursor, OprE Gln Ala Gly Ala (SEQ ID NO: 8) Putative phosphateLys Leu Lys Arg Leu Met Ala Ala Met Thr Phe Val Ala Ala Glybinding protein (SEQ ID NO: 9) Putative azurinPhe Ala Lys Leu Val Ala Val Ser Leu Leu Thr Leu Ala Ser GlyGln Leu Leu Ala (SEQ ID NO: 10) Putative periplasmicIle Lys Arg Asn Leu Leu Val Met Gly Leu Ala Val Leu Leu Serlipoprotein B precursor (SEQ ID NO: 11) Putative Lys-Arg-OrnGln Asn Tyr Lys Lys Phe Leu Leu Ala Ala Ala Val Ser Met Alabinding protein Phe Ser Ala Thr Ala Met Ala (SEQ ID NO: 12)Putative Fe(III) bindingMet Ile Arg Asp Asn Arg Leu Lys Thr Ser Leu Leu Arg Gly Leu proteinThr Leu Thr Leu Leu Ser Leu Thr Leu Leu Ser Pro Ala Ala His Ser(SEQ ID NO: 13)Western Analysis of the phoA Fusion Proteins to Detect Fusion Proteins

To analyze whether the fusion proteins were produced, Western analysiswith antibody to alkaline phosphatase was carried out on culturesseparated by centrifugation into a whole-cell fraction (cytoplasm andperiplasm) and a cell-free broth fraction. Of five strains for which thesite of insertion was determined, four (putative azurin, putativephosphate binding protein, putative periplasmic lipoprotein B, putativeFe(III) binding protein) produced a fusion protein of the expected size,and one (putative oprE protein) produced a protein about 40 kD smallerthan predicted, and one (putative Lys-Arg-Orn binding protein) produceda protein about 20 kD smaller than predicted.

Proteins were separated by SDS-PAGE and were transferred tonitrocellulose membrane at 40 V for one hour using the Xcell SureLock™Mini-Cell and XCell II TM Blot Module (Invitrogen). Western experimentswere performed using the instruction provided from SuperSignal WestHisProbe™ Kit (Pierce).

Construction, Expression, and Characterization of a pbp-hGH Fusion

The P. fluorescens phosphate binding protein secretion leader was fusedto the N-terminus of the mature domain of the human growth hormone (hGH)gene and tested for expression and secretion.

The pbp signal-sequence coding region was PCR amplified from a clone ofthe P. fluorescens pbp signal sequence as template, using sig_pbp for(gctctagaggaggtaacttatgaaactgaaacg (SEQ ID NO: 22)) and pbp_hgh(gggaatggttgggaaggccaccgcgttggc (SEQ ID NO: 23)) primers, thengel-purified. This resulted in production of an oligonucleotide fragmentcontaining the pbp signal peptide CDS and the coding sequence for the 5′end of the mature domain of hGH.

A cDNA encoding the human growth hormone was PCR-amplified from a humanpituitary cDNA library (Clontech, Palo Alto Calif.) using primersELVIfor (agagaactagtaaaaaggagaaatccatggctacaggctcccggacgtcc) (SEQ ID NO:6) and ELVIrev (agagactcgagtcattagaagccacagctgccctccac) (SEQ ID NO: 7),which were designed to amplify only the mature domain of hGH, and clonedinto pMYC1 803/SpeI XhoI, forming pDOW2400. The mature hGH gene wasamplified from pDOW2400, using primers pbp_hgh_revcomp(gccaacgcggtggccttcccaaccattccc) (SEQ ID NO: 14) and hgh_rev(agagactcgagtcattagaagccacagctgccctccacagagcggcac) (SEQ ID NO: 15), thenpurified with Strataprep columns (Stratagene) to remove primers andother reaction components. To make the polynucleotide encoding thepbp-hGH fusion, the two PCR reactions were combined and amplified againwith sig_pbp for and hgh_rev in order to link the two pieces. Theexpected 681 bp fragment was purified with Strataprep as above,restriction digested with XbaI and XhoI and ligated to dephosphorylatedpDOW1269/XhoISpeI to form pDOW 1323-10, placing pbp-hGH under control ofthe tac promoter in a vector analogous to pMYC1803, but with a pyrFselectable marker in place of a tetR tetracycline resistance markergene. The ligation mix was transformed into MB101 pyrF proC lacI^(QI).Inserts were sequenced by The Dow Chemical Company using the methoddescribed above. The DNA and amino acid sequence of this fusion ispresented in (FIG. 10) and (FIG. 11), respectively.

The resulting strains were tested first at the shake flask scale.Induced bands of the expected size for processed and unprocessed (22.2kDa and 24.5 kDa, respectively) were detected by SDS-PAGE. About half ofthe protein was processed (indicating localization to the periplasm),and of the processed about half was in the soluble fraction and half inthe insoluble fraction. Expression studies were scaled up to 20-Lbioreactors. Densitometry of the Coomassie-stained SDS-PAGE gels showedthat 18% of the total hGH produced was processed and soluble. The strainproduced 3.2 g/L of all forms of hGH; processed and soluble was 0.6 g/L.

Construction, Expression, and Characterization of pbp-scFv Fusion

The putative 24 amino acid signal sequence of phosphate binding protein(i.e. including Metl) was fused to the open reading frame of the gal2scFv gene (ga12) at the +2 amino acid (Ala) position. See FIG. 8 andFIG. 9. The signal sequence appears to be processed, indicatingsecretion to the periplasm. Moreover, there is secretion to the broth,in that protein was detected in the cell free culture supernatant.Surprisingly, fusion to the phosphate binding protein signal sequenceappears to improve expression of ga12 scFv in P. fluorescens. Withoutthe secretion signal fused at the amino terminus, expression of ga12scFv was not detectable.

Cloning of Gal2

PCR was performed using primers sigpbp for (above) and pbp_gal2SOE rev(ctgcacctgggcggccaccgcgtt (SEQ ID NO: 24)), which contains a reversecomplement of pbp_gal2SOE for (aaccgcggtggccgcccaggtgcag (SEQ ID NO:25)), and using a plasmid encoding the P. fluorescens pbp secretionsignal peptide as template. This resulted in production of anoligonucleotide fragment containing the pbp signal peptide codingsequence (CDS) and a CDS for the 5′ end of the gal2 single chainantibody (scAb or scFv).

PCR was performed using primers pbp_gal2SOE for and scFv2rev(acgcgtcgacttattaatggtg, (SEQ ID NO: 26) atgatggtgatgtgcggccgcacgtttgatc(SEQ ID NO: 27)), and using a ga12-encoding polynucleotide as template.This resulted in production of a polynucleotide fragment containing aCDS encoding the 3′end of the pbp signal peptide and the open readingframe (ORE) encoding ga12.

Reaction products were purified. About 15 ng of each was used as a“template” DNA in a further PCR reaction using primers sig_pbpfor andscFv2rev. This resulted in production of a nucleic acid fragment withthe pbp signal peptide CDS fused to the gal2 coding sequence.

The predicted -1 amino acid of the signal sequence (that is the lastamino acid prior to the proposed cleavage site) was fused to the +2amino acid of the ga12 scFv (Ala). The resulting fusion was cloned intothe P. fluorescens vector pMYC1803 under control of the Ptac promoter toproduce plasmid and pDOW1123 (pbp:gal2). The plasmid was transformedinto P. fluorescens strain MB 101 carrying plasmid pCN51-lacI (describedin U.S. application Ser. No. 10/994,138, filed Nov. 19, 2004.

Fusion of the Putative Phosphate Binding Protein Signal Sequence to gal2scFv

The phosphate binding protein signal sequence was fused to a singlechain antibody gene and tested for secretion to the periplasm and/or tothe culture supernatant.

TABLE 13 secreted Gal2 fermentation summary (*compared to BSA standards)E. coli P. fluorescens Pf/Ec Fermentation Time (hr) 8-9 50-70 8 Max hGHtitre (*g/L) 1.6 (0.8 9.3 (25% cv) 6 (12) processed) Dry biomass (g/L)18 (70) 4 hGH/biomass (% w/w) 8.9 (4.4 13 1.5 (3) processed)

The resulting strains were tested first at the shake flask scale.Induced bands of the expected size for unprocessed and processed gal2(29 kDa and 27 kDa) were detected via SDS-PAGE in the insoluble proteinfraction (data not shown). Expression studies were scaled up to 20 Lfermentation. Again, SDS-PAGE analysis showed that the majority of theinduced protein is found in the insoluble protein fraction.

The Western analysis also indicated that some processed ga12 is presentin the soluble protein fraction for pbp:gal2 (pDOW1123). Westernanalysis of periplasmic fractions prepared from strains carrying pDOW1123 (using the Epicentre periplast kit) showed the presence of solublegal2 protein.

Recombinant ga12 scFv was isolated from the cell extract of a shakeflask experiment using the Qiagen Ni-NTA protocol, then refolded asdescribed in P. Martineau et al., J Mol. Biol. 280:117-127 (1998). Thisantibody was found to be active against β-galactosidase in an ELISAassay.

Example 5

Expression of bovine γ-IFN in the Cytoplasm

Pseudomonas Fluorescens Host Cells and Expression Systems

Production strain MB324 was used for transformation experiments andplasmid pHYC1803 was used for subcloning experiments. The Bacillusthuringiensis BuiBui insert of the vector was excised with restrictionenzyme SpeI and XhoI prior to insertion of the bovine IFN-γ (BGI) gene.The published nucleotide sequence of BGI was obtained from GenBank usingSeqWeb software. The sequence to be synthesized was modified to excludethe signal sequence and include ribosome binding, SpeI and XhoIrestriction sites.

Subcloning of Interferon Genes

Conical tubes (50 mL) containing 5-mL L-broth (LB) were inoculated withice chips from frozen glycerol stock cultures of P. fluorescens MB324.The cultures were incubated in a rotary shaker overnight at 300 rpm and30° C. 0.75 mL from each culture was used to inoculate 50 mL of LB in250-mL side-baffled flasks. The cultures were shaken for two hours at300 rpm and 30° C. and grown to an A600 (absorbance at 600 nM) of 0.2 to0.3. Cultures were then cooled on ice and pelleted by centrifugation at3000 rpm. Pelleted materials was washed with cold, sterile, distilledwater three times and the pellets were re-suspended in water.

The cell suspensions (about 100 μL each) were added to electroporationcuvettes, mixed with 10 μL of either interferon gene or control ligationmixtures; re-suspended cells were electroporated with a BioRadGenePulser in 0.2 cm cuvettes at 200 ohms, 25 μF and 2.25 kV and“pulsed” at time-constants between 4.6 and 4.8.

One-mL of LB was added to each sample, and the liquid was transferred toiced 2059 Falcon tubes. The tubes were loosely capped, shaken for twohours at 280 rpm and 30° C. 100 μL, to 200 μL, aliquots were plated onL-broth-tetracycline (LB-tetracycline) (30 μg/mL) agar and incubated at30° C. overnight. One colony from each of two 100 μL platings and twocolonies from a 200 μL, plating were randomly selected and used toinoculate 50 mL conical tubes with LB-tetracycline broth, as describedabove. Samples of the resulting cultures were mixed with sterileglycerol (1.0 mL culture plus 0.25 mL 75% glycerol) and stored at −70°C. The remaining culture (1.8 mL) was centrifuged for 10 minutes in a 2mL Eppendorf tube. The pellets were re-suspended in 0.5 mL of Qiagen P1solution, followed by gentle inversion six-times with 0.5 mL P2solution.

Within about five minutes, the sample was re-inverted six times with N3solution and iced. The chilled sample was centrifuged for ten minutes,carefully separated from the pellet and surface scum, and the resultingsupernatant liquid (about 1.5 mL) was transferred to a fresh Eppendorftube. The sample was further purified with a Qiagen spin column andcollection tube by spin-loading the entire 1.5 mL sample onto the columnwith two, 30 second, 14000 RPM (14 K) spins of about 0.7 mL to 0.8 mLaliquots. The spin-column was washed with 0.62 mL Qiagen PB and 0.85 mLPE, with a final spin of 90 seconds. The column was transferred to a newEppendorf tube, eluted for 1 minute with 50 μL Tris-EDTA, and spun forone minute at 14 K The eluent was transferred to a new Eppendorf tubeand stored at −20° C. The resulting mini-preps were digested with XhoIand SpeI and analyzed by agarose-gel electrophoresis.

Expression and Quantitation of Interferon Protein

Based on these results, one clone of MR324 with an IFN-γ insert wasselected for expression analysis. P. fluorescens strains MR843 and MR837were used as interferon-negative controls. LB-tetracycline seed-flaskswere grown to A600 0.15 to 0.5 and normalized to 0.15 for 2% dilutioninto 1-liter shake flasks containing 200-mL tetracycline productionmedium. P. fluorescens cells were grown to approximately A600 0.4 at 30°C. with rotary shaking for 24 hours. The cells were induced with 0.6 mLof 100 mM IPTG +5 mL 40% MSG for an additional 48 hours. The cells wereexamined microscopically for general appearance and inclusion bodyformation.

Fifty-mL samples were taken and stored at 4° C. in conical tubes foranalysis of expression by sodium dodecylsulfate polyacrylamide-gelelectrophoresis (SDS-PAGE.) A total of 100 μL was centrifuged for fiveminutes at 14 K to pellet the cells. Pellets were re-suspended in 100 μLLaemmli buffer and boiled for 3 minutes, and supernatant samples werediluted 1:1 with Laemmli buffer prior to being boiled. Ten μL of boiledsample were mixed with 30 μL of fresh Laemmli buffer and boiled for anadditional 3-minutes. The preparations were frozen overnight, thawed thefollowing day, heated to 70° C. for five minutes, loaded (10 μL each)into the wells of a 12-lane, 15% BioRad gel, and electrophoresed withBioRad running buffer. The electrophoresis ran for 20 minutes at 50volts followed by 1 hour 20-minutes at 75 volts. After the run, the gelswere washed in distilled water three times for five minutes each andstained with BioRad's BioSafe stain for 1.5 hours. The stained gels werede-stained in distilled water with one change after one hour.Quantitation was accomplished with an MD densitometer by comparing theCoomassie Blue intensity of the samples to interferon-minus controls anda BSA protein standard.

The BuiBui toxin gene was replaced with the bovine gamma-interferon(BGI) gene at the SpeI and XhoI sites of pMYC1803. All the transformantsselected had the desired interferon insert, as verified first byagarose-gel electrophoresis, and then by sequencing the inserted DNA.One clone of the DnaK, chaperonin containing strain of P. fluorescens,MB324, was selected for further study.

A major band of protein was observed at the molecular weight expectedfor BGI and expression of BGI in Pseudomonas was about 40% of totalcellular protein. Identity of this major band with authentic BGI wasconfirmed by purification of the protein contained in the major band,coupled with bioassays of the purified product. With the optimization ofexpression and high-density fermentation achievable with Pseudomonas,interferon production of greater than 1000 Kg can be attained in asingle fermentation production run.

Solubility Assay

A 0.975 mL volume of P. fluorescens culture was centrifuged in amicrofuge for 5 minutes at 14,000 RPM. The supernatant liquid wasdecanted and the cells were resuspended in lysis buffer up to thestarting volume. (Lysis buffer: Tris HCl, 50 mM, pH 7.5 final; NaCl, 200mM; glycerol, 5% v/v; EDTA, 20 mM; Triton X-100, 5% v/v; and, addedlast, DTT, 1 mM) Screw-cap microfuge tubes (2mL) were filled about ¾full with 0.1 mm glass beads and topped off with cell suspension. Thetubes were given a quick shake to mix the beads and remove air bubbles,and further filled to the top with cell suspension. The tubes werecapped, sealed, and inserted into a BioSpec mini bead-beater for 60seconds at 5000 rpm. The samples were kept on ice between beatings andbeat 3 to 5 times until about 90% of the cells were lysed. Cell lysiswas observed by microscopic observation. A volume of 0.025 mL of thelysed cell preparation was pipetted from each tube, minus beads, intonew microfuge tubes and centrifuged for 5 minutes. The supernatantfraction was carefully transferred to another tube with 0.075 mL LSB,and 0.100 mL LSB was added to the pellet fraction. The supernatant andpellet fractions were re-suspended with a Vortex stirrer, the tubes werecapped, placed in a boiling water bath for five minutes, and 0.005 mL to0.010 mL aliquots of the fractions SDS PAGE were analyzed. Assessment ofexpressed BGI protein solubility in Pseudomonas, using either aFrench-Press or a BioSpec Mini Bead-Beater produced equivalent results.

The solubility of BGI in Pseudomonas cells was tested and indicated thatmost, if not all, of the BGI remained in soluble form. To do thesesolubility-tests, viable unamended Pseudomonas cells were broken in aFrench Press (or mini-bead beater), and centrifuged to separate celldebris and any inclusion bodies from soluble proteins. SDS gels of thesetwo fractions indicated that BGI was retained in the soluble portion,whereas BAI (bovine α-interferon), a marker in this example that hadbeen cloned and expressed for another experiment, occurred primarily inthe insoluble fraction. Furthermore, unlike BGI, BAI formed largeinclusions in Pseudomonas, which were highly visible underphase-contrast microscopy.

SDS-PAGE analysis of French-pressed P. fluorescens cultures containingboth BAI and BGI were conducted. Pseudomonas cells were ruptured in aFrench Press and centrifuged at 16000 g for five minutes. Supernatantsamples showed a single, major band (about 17 kDa) of soluble BGI withno BAI visible. Pelleted samples showed a major band (about 18 kDa) ofinsoluble BAI together with small amounts of contaminating soluble BGI.The contamination appears to be due to spillover from the supernatantfraction and unlysed cells.

Both the amount and activity of BGI in Pseudomonas cells were high. Asillustrated in the examples, P. fluorescens is a good biofactory,capable of producing up to 40% or more of total cell protein asrecombinant protein, such as interferon and the cells produce activeprotein.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

What is claimed is:
 1. A process for increasing expression of arecombinant mammalian protein comprising: a. transforming a Pseudomonasfluorescens host cell with a nucleic acid encoding a recombinantmammalian protein; and b. growing the cell under conditions that allowexpression of the recombinant mammalian protein; wherein the protein isexpressed at an increased level when compared to an expression level ofthe protein under substantially comparable conditions in an E. coliexpression system.
 2. The process of claim 1, further comprisingisolating the recombinant mammalian protein.
 3. The process of claim 2,further comprising substantially purifying the recombinant mammalianprotein.
 4. The process of claim 1, wherein the recombinant mammalianprotein is present in the host cell in a soluble form.
 5. The process ofclaim 1, wherein the recombinant mammalian protein is present in thecell in an insoluble form.
 6. The process of claim 1, wherein therecombinant mammalian protein is present in the cell in an active form.7. The process of claim 1, wherein the recombinant mammalian protein isa human peptide.
 8. The process of claim 1 wherein the recombinantprotein is produced as more than about 5% total cell protein.
 9. Theprocess of claim 1 wherein the recombinant protein is produced as morethan about 10% total cell protein.
 10. The process of claim 1 whereinthe recombinant protein is produced at a concentration of at least 10g/L.
 11. The process of claim 1 wherein the recombinant protein isproduced at a concentration of at least 20 g/L.
 12. The process of claim1 wherein the recombinant protein is produced at a concentration of atleast 40 g/L.
 13. A process for producing a recombinant mammalianprotein in a Pseudomonas fluorescens host cell comprising: a.transforming a host cell with a nucleic acid encoding a recombinantmammalian protein; b. growing the cell under conditions that allowexpression of the recombinant mammalian protein; and c. isolating therecombinant mammalian protein.
 14. The process of claim 13, furthercomprising substantially purifying the recombinant mammalian protein.15. The process of claim 13, wherein the recombinant mammalian proteinis present in the host cell in a soluble form.
 16. The process of claim13, wherein the recombinant mammalian protein is present in the hostcell in an insoluble form.
 17. The process of claim 13, wherein therecombinant mammalian protein is present in the host cell in an activeform.
 18. The process of claim 13, wherein the recombinant mammalianprotein is a human peptide.
 19. The process of claim 13, wherein therecombinant mammalian protein has a mass of between at least about 1 kDand about 500 kD.
 20. The process of claim 19, wherein the recombinantmammalian protein has a mass of greater than about 30 kD.
 21. Theprocess of claim 13 wherein the recombinant protein is produced as morethan about 5% total cell protein.
 22. The process of claim 13 whereinthe recombinant protein is produced as more than about 10% total cellprotein.
 23. The process of claim 13 wherein the recombinant protein isproduced at a concentration of at least 10 g/L.
 24. The process of claim13 wherein the recombinant protein is produced at a concentration of atleast 20g/L.
 25. The process of claim 13 wherein the recombinant proteinis produced at a concentration of at least 40 g/L.
 26. A process forproducing a recombinant human protein in a host cell comprising: a.transforming a host cell with a nucleic acid encoding a recombinanthuman peptide; and b. growing the cell under conditions that allowexpression of the recombinant human peptide; wherein the host cell isPseudomonas fluorescens.
 27. The process of claim 26, wherein therecombinant human protein is present in soluble form in the host cell.28. The process of claim 26, wherein the recombinant human protein ispresent in an active form in the host cell.
 29. A Pseudomonasfluorescens cell comprising a nucleic acid encoding a recombinant humanpeptide.
 30. The cell of claim 29, wherein the recombinant human peptideis expressed.